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Tiskratok W, Chuinsiri N, Limraksasin P, Kyawsoewin M, Jitprasertwong P. Extracellular Matrix Stiffness: Mechanotransduction and Mechanobiological Response-Driven Strategies for Biomedical Applications Targeting Fibroblast Inflammation. Polymers (Basel) 2025; 17:822. [PMID: 40292716 PMCID: PMC11946729 DOI: 10.3390/polym17060822] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2025] [Revised: 03/13/2025] [Accepted: 03/19/2025] [Indexed: 04/30/2025] Open
Abstract
The extracellular matrix (ECM) is a dynamic network providing mechanical and biochemical cues that regulate cellular behavior. ECM stiffness critically influences fibroblasts, the primary ECM producers, particularly in inflammation and fibrosis. This review explores the role of ECM stiffness in fibroblast-driven inflammation and tissue remodeling, focusing on the physicochemical and biological mechanisms involved. Engineered materials, hydrogels, and polydimethylsiloxane (PDMS) are highlighted for replicating tissue-specific stiffness, enabling precise control over cell-matrix interactions. The surface functionalization of substrate materials, including collagen, polydopamine, and fibronectin, enhances bioactivity and fibroblast adhesion. Key mechanotransduction pathways, such as integrin signaling and YAP/TAZ activation, are related to regulating fibroblast behaviors and inflammatory responses. The role of fibroblasts in driving chronic inflammatory diseases emphasizes their therapeutic potentials. Advances in ECM-modifying strategies, including tunable biomaterials and hydrogel-based therapies, are explored for applications in tissue engineering, drug delivery, anti-inflammatory treatments, and diagnostic tools for the accurate diagnosis and prognosis of ECM stiffness-related inflammatory diseases. This review integrates mechanobiology with biomedical innovations, providing a comprehensive prognosis of fibroblast responses to ECM stiffness and outlining future directions for targeted therapies.
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Affiliation(s)
- Watcharaphol Tiskratok
- Institute of Dentistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (N.C.); (P.J.)
- Oral Health Centre, Suranaree University of Technology Hospital, Nakhon Ratchasima 30000, Thailand
| | - Nontawat Chuinsiri
- Institute of Dentistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (N.C.); (P.J.)
- Oral Health Centre, Suranaree University of Technology Hospital, Nakhon Ratchasima 30000, Thailand
| | - Phoonsuk Limraksasin
- Center of Excellence for Dental Stem Cell Biology, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; (P.L.); (M.K.)
| | - Maythwe Kyawsoewin
- Center of Excellence for Dental Stem Cell Biology, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; (P.L.); (M.K.)
| | - Paiboon Jitprasertwong
- Institute of Dentistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; (N.C.); (P.J.)
- Oral Health Centre, Suranaree University of Technology Hospital, Nakhon Ratchasima 30000, Thailand
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Jaganathan A, Toth J, Chen X, Basir R, Pieuchot L, Shen Y, Reinhart-King C, Shenoy VB. Mechano-metabolism of metastatic breast cancer cells in 2D and 3D microenvironments. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.30.591879. [PMID: 38746096 PMCID: PMC11092625 DOI: 10.1101/2024.04.30.591879] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
Cells regulate their shape and metabolic activity in response to the mechano-chemical properties of their microenvironment. To elucidate the impact of matrix stiffness and ligand density on the bioenergetics of mesenchymal cells, we developed a nonequilibrium, active chemo-mechanical model that accounts for the mechanical energy of the cell and matrix, chemical energy from ATP hydrolysis, interfacial energy, and mechano-sensitive regulation of stress fiber assembly through signaling. By integrating the kinetics and energetics of these processes, we define the cell "metabolic potential" that, when minimized, provides testable predictions of cell contractility, shape, and ATP consumption. Specifically, we show that the morphology of MDA-MB-231 breast cancer cells in 3D collagen changes from spherical to elongated to spherical with increasing matrix stiffness, which is consistent with experimental observations. On 2D hydrogels, our model predicts a hemispherical-to-spindle-to-disc shape transition with increasing gel stiffness. In both cases, we show that these shape transitions emerge from competition between the energy of ATP hydrolysis associated with increased contractility that drives cell elongation and the interfacial energy that favors a rounded shape. Furthermore, our model can predict how increased energy demand in stiffer microenvironments is met by AMPK activation, which is confirmed experimentally in both 2D and 3D microenvironments and found to correlate with the upregulation of mitochondrial potential, glucose uptake, and ATP levels, as well as provide estimates of changes in intracellular adenosine nucleotide concentrations with changing environmental stiffness. Overall, we present a framework for relating adherent cell energy levels and contractility through biochemical regulation of underlying physical processes. Statement of Significance Increasing evidence indicates that cellular metabolism is regulated by mechanical cues from the extracellular environment. Forces transmitted from the microenvironment activate mechanotransduction pathways in the cell, which trigger a cascade of biochemical events that impact cytoskeletal tension, cellular morphology and energy budget available to the cell. Using a nonequilibrium free energy-based theory, we can predict the ATP consumption, contractility, and shape of mesenchymal cancer cells, as well as how cells regulate energy levels dependent on the mechanosensitive metabolic regulator AMPK. The insights from our model can be used to understand the mechanosensitive regulation of metabolism during metastasis and tumor progression, during which cells experience dynamic changes in their microenvironment and metabolic state.
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Alisafaei F, Mandal K, Saldanha R, Swoger M, Yang H, Shi X, Guo M, Hehnly H, Castañeda CA, Janmey PA, Patteson AE, Shenoy VB. Vimentin is a key regulator of cell mechanosensing through opposite actions on actomyosin and microtubule networks. Commun Biol 2024; 7:658. [PMID: 38811770 PMCID: PMC11137025 DOI: 10.1038/s42003-024-06366-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Accepted: 05/21/2024] [Indexed: 05/31/2024] Open
Abstract
The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.
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Affiliation(s)
- Farid Alisafaei
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Department of Mechanical and Industrial Engineering, New Jersey Institute of Technology, Newark, NJ, 07102, USA
| | - Kalpana Mandal
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Institute for Medicine and Engineering, University of Pennsylvania, 3340 Smith Walk, Philadelphia, PA, 19104, USA
| | - Renita Saldanha
- Physics Department, Syracuse University, Syracuse, NY, 13244, USA
- BioInspired Institute, Syracuse University, Syracuse, NY, 13244, USA
| | - Maxx Swoger
- Physics Department, Syracuse University, Syracuse, NY, 13244, USA
- BioInspired Institute, Syracuse University, Syracuse, NY, 13244, USA
| | - Haiqian Yang
- Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Xuechen Shi
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Institute for Medicine and Engineering, University of Pennsylvania, 3340 Smith Walk, Philadelphia, PA, 19104, USA
| | - Ming Guo
- Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
| | - Heidi Hehnly
- Department of Biology, Syracuse University, Syracuse, NY, 13244, USA
| | - Carlos A Castañeda
- Departments of Biology and Chemistry, Syracuse University, Syracuse, NY, 13244, USA
- Interdisciplinary Neuroscience Program, Syracuse University, Syracuse, NY, 13244, USA
| | - Paul A Janmey
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, 19104, USA
- Institute for Medicine and Engineering, University of Pennsylvania, 3340 Smith Walk, Philadelphia, PA, 19104, USA
- Departments of Physiology, and Physics & Astronomy, University of Pennsylvania, Philadelphia, PA, 19104, USA
| | - Alison E Patteson
- Physics Department, Syracuse University, Syracuse, NY, 13244, USA
- BioInspired Institute, Syracuse University, Syracuse, NY, 13244, USA
| | - Vivek B Shenoy
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, 19104, USA.
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Hu S, Meng K, Wang T, Qu R, Wang B, Xi Y, Yu T, Yuan Z, Cai Z, Tian Y, Zeng C, Wang X, Zou W, Fu X, Li L. Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy. Biomark Res 2024; 12:40. [PMID: 38637902 PMCID: PMC11027539 DOI: 10.1186/s40364-024-00586-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Accepted: 03/29/2024] [Indexed: 04/20/2024] Open
Abstract
BACKGROUND IL-15 plays a vital role in enhancing NK cell- and T-cell-mediated antitumor immune responses; however, the direct effect of IL-15 on tumor cells has not been fully elucidated. Herein, we investigated the effect of IL-15 on lung adenocarcinoma cells. METHODS Silencing and overexpression techniques were used to modify endogenous IL-15 expression in tumor cells. Transwell assays were used to assess tumor cell migration and invasion; a live-cell analysis system was used to evaluate cell motility; cellular morphological changes were quantified by confocal fluorescence microscopy; the molecular mechanisms underlying the effect of IL-15 on tumor cells were analyzed by western blotting; and RhoA and Cdc42 activities were evaluated by a pulldown assay. NCG and C57BL/6 mouse models were used to evaluate the functions of IL-15 in vivo. RESULTS Cancer cell-intrinsic IL-15 promoted cell motility and migration in vitro and metastasis in vivo via activation of the AKT-mTORC1 pathway; however, exogenous IL-15 inhibited cell motility and migration via suppression of the RhoA-MLC2 axis. Mechanistic analysis revealed that both the intracellular and extracellular IL-15-mediated effects required the expression of IL-15Rα by tumor cells. Detailed analyses revealed that the IL-2/IL-15Rβ and IL-2Rγ chains were undetected in the complex formed by intracellular IL-15 and IL-15Rα. However, when exogenous IL-15 engaged tumor cells, a complex containing the IL-15Rα, IL-2/IL-15Rβ, and IL-2Rγ chains was formed, indicating that the differential actions of intracellular and extracellular IL-15 on tumor cells might be caused by their distinctive modes of IL-15 receptor engagement. Using a Lewis lung carcinoma (LLC) metastasis model, we showed that although IL-15 overexpression facilitated the lung metastasis of LLC cells, IL-15-overexpressing LLC tumors were more sensitive to anti-PD-L1 therapy than were IL-15-wild-type LLC tumors via an enhanced antitumor immune response, as evidenced by their increased CD8+ T-cell infiltration compared to that of their counterparts. CONCLUSIONS Cancer cell-intrinsic IL-15 and exogenous IL-15 differentially regulate cell motility and migration. Thus, cancer cell-intrinsic IL-15 acts as a double-edged sword in tumor progression. Additionally, high levels of IL-15 expressed by tumor cells might improve the responsiveness of tumors to immunotherapies.
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Affiliation(s)
- Shaojie Hu
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Kelin Meng
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Tianlai Wang
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Rirong Qu
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Boyu Wang
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Yu Xi
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Taiyan Yu
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Zhiwei Yuan
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Zihao Cai
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Yitao Tian
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Chenxi Zeng
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Xue Wang
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Wenbin Zou
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China
| | - Xiangning Fu
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China.
| | - Lequn Li
- Thoracic Surgery Laboratory, Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie Fang Avenue, 430030, Wuhan, Hubei, China.
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Chi Z, Wang Q, Tong L, Qiu J, Yang F, Guo Q, Li W, Zheng J, Chen Z. Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation. Cell Biol Int 2024; 48:174-189. [PMID: 37853939 DOI: 10.1002/cbin.12096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Revised: 09/05/2023] [Accepted: 09/30/2023] [Indexed: 10/20/2023]
Abstract
Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.
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Affiliation(s)
- Zengpeng Chi
- Department of Stomatology, Qingdao West Coast New District Central Hospital, Qingdao, China
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Qimin Wang
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Lei Tong
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Jing Qiu
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Fang Yang
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Qingyuan Guo
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Wenjian Li
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
| | - Jiawei Zheng
- Department of Oromaxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Zhenggang Chen
- Department of Stomatology, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, China
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Wang Y, Troughton LD, Xu F, Chatterjee A, Ding C, Zhao H, Cifuentes LP, Wagner RB, Wang T, Tan S, Chen J, Li L, Umulis D, Kuang S, Suter DM, Yuan C, Chan D, Huang F, Oakes PW, Deng Q. Atypical peripheral actin band formation via overactivation of RhoA and nonmuscle myosin II in mitofusin 2-deficient cells. eLife 2023; 12:e88828. [PMID: 37724949 PMCID: PMC10550287 DOI: 10.7554/elife.88828] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Accepted: 09/19/2023] [Indexed: 09/21/2023] Open
Abstract
Cell spreading and migration play central roles in many physiological and pathophysiological processes. We have previously shown that MFN2 regulates the migration of human neutrophil-like cells via suppressing Rac activation. Here, we show that in mouse embryonic fibroblasts, MFN2 suppresses RhoA activation and supports cell polarization. After initial spreading, the wild-type cells polarize and migrate, whereas the Mfn2-/- cells maintain a circular shape. Increased cytosolic Ca2+ resulting from the loss of Mfn2 is directly responsible for this phenotype, which can be rescued by expressing an artificial tether to bring mitochondria and endoplasmic reticulum to close vicinity. Elevated cytosolic Ca2+ activates Ca2+/calmodulin-dependent protein kinase II, RhoA, and myosin light-chain kinase, causing an overactivation of nonmuscle myosin II, leading to a formation of a prominent F-actin ring at the cell periphery and increased cell contractility. The peripheral actin band alters cell physics and is dependent on substrate rigidity. Our results provide a novel molecular basis to understand how MFN2 regulates distinct signaling pathways in different cells and tissue environments, which is instrumental in understanding and treating MFN2-related diseases.
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Affiliation(s)
- Yueyang Wang
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Lee D Troughton
- Cell and Molecular Physiology, Loyola University ChicagoChicagoUnited States
| | - Fan Xu
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
- Advanced Research Institute of Multidisciplinary Science, Beijing Institute of TechnologyBeijingChina
| | - Aritra Chatterjee
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Chang Ding
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Han Zhao
- Davidson School of Chemical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Laura P Cifuentes
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Ryan B Wagner
- School of Mechanical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Tianqi Wang
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Shelly Tan
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Jingjuan Chen
- Department of Animal Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Linlin Li
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - David Umulis
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
- Department of Agricultural and Biological Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Shihuan Kuang
- Department of Animal Sciences, Purdue University West LafayetteWest LafayetteUnited States
| | - Daniel M Suter
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
- Purdue Institute for Integrative Neuroscience, Purdue University West LafayetteWest LafayetteUnited States
- Purdue Institute for Inflammation, Immunology & Infectious Disease, Purdue University West LafayetteWest LafayetteUnited States
| | - Chongli Yuan
- Davidson School of Chemical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Deva Chan
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Fang Huang
- Weldon School of Biomedical Engineering, Purdue University West LafayetteWest LafayetteUnited States
| | - Patrick W Oakes
- Cell and Molecular Physiology, Loyola University ChicagoChicagoUnited States
| | - Qing Deng
- Department of Biological Sciences, Purdue University West LafayetteWest LafayetteUnited States
- Purdue Institute for Inflammation, Immunology & Infectious Disease, Purdue University West LafayetteWest LafayetteUnited States
- Purdue University Center for Cancer Research, Purdue University West LafayetteWest LafayetteUnited States
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Sharma A, Rahman G, Gorelik J, Bhargava A. Voltage-Gated T-Type Calcium Channel Modulation by Kinases and Phosphatases: The Old Ones, the New Ones, and the Missing Ones. Cells 2023; 12:461. [PMID: 36766802 PMCID: PMC9913649 DOI: 10.3390/cells12030461] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 01/14/2023] [Accepted: 01/29/2023] [Indexed: 02/04/2023] Open
Abstract
Calcium (Ca2+) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet.
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Affiliation(s)
- Ankush Sharma
- Department of Biotechnology, Indian Institute of Technology Hyderabad (IITH), Kandi 502284, Telangana, India
| | - Ghazala Rahman
- Department of Biotechnology, Indian Institute of Technology Hyderabad (IITH), Kandi 502284, Telangana, India
| | - Julia Gorelik
- National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London W12 0NN, UK
| | - Anamika Bhargava
- Department of Biotechnology, Indian Institute of Technology Hyderabad (IITH), Kandi 502284, Telangana, India
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Basu A, Paul MK, Weiss S. The actin cytoskeleton: Morphological changes in pre- and fully developed lung cancer. BIOPHYSICS REVIEWS 2022; 3:041304. [PMID: 38505516 PMCID: PMC10903407 DOI: 10.1063/5.0096188] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/15/2022] [Accepted: 12/09/2022] [Indexed: 03/21/2024]
Abstract
Actin, a primary component of the cell cytoskeleton can have multiple isoforms, each of which can have specific properties uniquely suited for their purpose. These monomers are then bound together to form polymeric filaments utilizing adenosine triphosphate hydrolysis as a source of energy. Proteins, such as Arp2/3, VASP, formin, profilin, and cofilin, serve important roles in the polymerization process. These filaments can further be linked to form stress fibers by proteins called actin-binding proteins, such as α-actinin, myosin, fascin, filamin, zyxin, and epsin. These stress fibers are responsible for mechanotransduction, maintaining cell shape, cell motility, and intracellular cargo transport. Cancer metastasis, specifically epithelial mesenchymal transition (EMT), which is one of the key steps of the process, is accompanied by the formation of thick stress fibers through the Rho-associated protein kinase, MAPK/ERK, and Wnt pathways. Recently, with the advent of "field cancerization," pre-malignant cells have also been demonstrated to possess stress fibers and related cytoskeletal features. Analytical methods ranging from western blot and RNA-sequencing to cryo-EM and fluorescent imaging have been employed to understand the structure and dynamics of actin and related proteins including polymerization/depolymerization. More recent methods involve quantifying properties of the actin cytoskeleton from fluorescent images and utilizing them to study biological processes, such as EMT. These image analysis approaches exploit the fact that filaments have a unique structure (curvilinear) compared to the noise or other artifacts to separate them. Line segments are extracted from these filament images that have assigned lengths and orientations. Coupling such methods with statistical analysis has resulted in development of a new reporter for EMT in lung cancer cells as well as their drug responses.
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Affiliation(s)
| | | | - Shimon Weiss
- Author to whom correspondence should be addressed:
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Effects of Electrical Stimulation on the Signal Transduction-Related Proteins, c-Src and Focal Adhesion Kinase, in Fibroblasts. Life (Basel) 2022; 12:life12040531. [PMID: 35455022 PMCID: PMC9024655 DOI: 10.3390/life12040531] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2022] [Revised: 03/28/2022] [Accepted: 04/02/2022] [Indexed: 11/29/2022] Open
Abstract
Electrical stimulation of the skin and muscles, e.g., in the fields of rehabilitation medicine and acupuncture, is known to locally increase blood flow and metabolism, and thus have beneficial health effects. However, little is known about the changes in cellular morphology or regulation of the localization of specific proteins in response to electrical stimuli. The present study was performed to examine the effects of electrical stimulation on the cytoskeletal system of cultured fibroblasts. Following application of electrical stimulation to cultured fibroblastic cells for a period of about 2 h, the stress fibers in the cells became thicker and the cells showed a contracted appearance. Cells were subjected to periodic electrical stimulation for 0 (unstimulated control), 2, 5, or 20 h. The stress fibers showed an increase in thickness within 2 h, and became gradually thicker until 20 h. In addition, the focal adhesions and stress fibers were enlarged after 2 h of continuous stimulation, and both stress fibers and focal adhesions became larger and thicker after 20 h of periodic stimulation. Cells showed increased staining of focal adhesions with anti-phosphotyrosine antibody (PY-20) after electrical stimulation. Cells also showed increased staining of tyrosine-phosphorylated focal adhesion kinase (FAK) (pY397) and tyrosine-phosphorylated c-Src (pY418), indicating that electrical stimulation affected signal transduction-related proteins.
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10
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Ma J, Bi L, Spurlin J, Lwigale P. Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development. eLife 2022; 11:74307. [PMID: 35238772 PMCID: PMC8916771 DOI: 10.7554/elife.74307] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Accepted: 03/02/2022] [Indexed: 11/19/2022] Open
Abstract
During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-seq analysis identified upregulation of nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues, including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itgα8) receptor. Knockdown of either Npnt or Itgα8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD-binding sites did not affect corneal thickness. Neither the knockdown nor augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itgα8, focal adhesion kinase, and Rho kinase. Combined, these data suggest that Npnt augments cell migration into the presumptive cornea extracellular matrix by functioning as a substrate for Itgα8-positive pNC cells.
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Affiliation(s)
- Justin Ma
- Department of Biosciences, Rice University, Houston, United States
| | - Lian Bi
- Department of Biosciences, Rice University, Houston, United States
| | - James Spurlin
- Department of Biosciences, Rice University, Houston, United States
| | - Peter Lwigale
- Department of Biosciences, Rice University, Houston, United States
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11
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Peng X, Huang Y, Alisafaei F. Cytoskeleton-mediated alterations of nuclear mechanics by extracellular mechanical signals. Biophys J 2022; 121:1-3. [PMID: 35000687 PMCID: PMC8758414 DOI: 10.1016/j.bpj.2021.12.017] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 11/29/2021] [Accepted: 12/09/2021] [Indexed: 01/07/2023] Open
Affiliation(s)
- Xiangjun Peng
- NSF Science and Technology Center for Engineering Mechanobiology,Department of Biomedical Engineering, Washington University, St. Louis, Missouri
| | - Yuxuan Huang
- NSF Science and Technology Center for Engineering Mechanobiology,Department of Mechanical Engineering and Materials Science, Washington University, St. Louis, Missouri
| | - Farid Alisafaei
- NSF Science and Technology Center for Engineering Mechanobiology,Department of Mechanical and Industrial Engineering, New Jersey Institute of Technology, Newark, New Jersey,Corresponding author
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12
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Petzold J, Gentleman E. Intrinsic Mechanical Cues and Their Impact on Stem Cells and Embryogenesis. Front Cell Dev Biol 2021; 9:761871. [PMID: 34820380 PMCID: PMC8606660 DOI: 10.3389/fcell.2021.761871] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Accepted: 10/14/2021] [Indexed: 12/25/2022] Open
Abstract
Although understanding how soluble cues direct cellular processes revolutionised the study of cell biology in the second half of the 20th century, over the last two decades, new insights into how mechanical cues similarly impact cell fate decisions has gained momentum. During development, extrinsic cues such as fluid flow, shear stress and compressive forces are essential for normal embryogenesis to proceed. Indeed, both adult and embryonic stem cells can respond to applied forces, but they can also detect intrinsic mechanical cues from their surrounding environment, such as the stiffness of the extracellular matrix, which impacts differentiation and morphogenesis. Cells can detect changes in their mechanical environment using cell surface receptors such as integrins and focal adhesions. Moreover, dynamic rearrangements of the cytoskeleton have been identified as a key means by which forces are transmitted from the extracellular matrix to the cell and vice versa. Although we have some understanding of the downstream mechanisms whereby mechanical cues are translated into changes in cell behaviour, many of the signalling pathways remain to be defined. This review discusses the importance of intrinsic mechanical cues on adult cell fate decisions, the emerging roles of cell surface mechano-sensors and the cytoskeleton in enabling cells to sense its microenvironment, and the role of intracellular signalling in translating mechanical cues into transcriptional outputs. In addition, the contribution of mechanical cues to fundamental processes during embryogenesis such as apical constriction and convergent extension is discussed. The continued development of tools to measure the biomechanical properties of soft tissues in vivo is likely to uncover currently underestimated contributions of these cues to adult stem cell fate decisions and embryogenesis, and may inform on regenerative strategies for tissue repair.
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Affiliation(s)
- Jonna Petzold
- Centre for Craniofacial and Regenerative Biology, King's College London, London, United Kingdom
| | - Eileen Gentleman
- Centre for Craniofacial and Regenerative Biology, King's College London, London, United Kingdom
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13
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Starostina I, Jang YK, Kim HS, Suh JS, Ahn SH, Choi GH, Suk M, Kim TJ. Distinct calcium regulation of TRPM7 mechanosensitive channels at plasma membrane microdomains visualized by FRET-based single cell imaging. Sci Rep 2021; 11:17893. [PMID: 34504177 PMCID: PMC8429465 DOI: 10.1038/s41598-021-97326-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Accepted: 08/17/2021] [Indexed: 11/09/2022] Open
Abstract
Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca2+ channel, plays a crucial role in intracellular Ca2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca2+ influx at plasma membrane microdomains. Using two different types of Ca2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels.
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Affiliation(s)
- Irina Starostina
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea
| | - Yoon-Kwan Jang
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea
| | - Heon-Su Kim
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea
| | - Jung-Soo Suh
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea
| | - Sang-Hyun Ahn
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea
| | - Gyu-Ho Choi
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea.,Department of Biological Sciences, Pusan National University, Pusan, 46241, Republic of Korea
| | - Myungeun Suk
- Department of Mechanical Engineering, Dong-Eui University, Pusan, 47340, Republic of Korea.
| | - Tae-Jin Kim
- Department of Integrated Biological Science, Pusan National University, Pusan, 46241, Republic of Korea. .,Department of Biological Sciences, Pusan National University, Pusan, 46241, Republic of Korea.
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14
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Singhatanadgit W, Hankamolsiri W, Janvikul W. Geranylgeraniol prevents zoledronic acid-mediated reduction of viable mesenchymal stem cells via induction of Rho-dependent YAP activation. ROYAL SOCIETY OPEN SCIENCE 2021; 8:202066. [PMID: 34113452 PMCID: PMC8187992 DOI: 10.1098/rsos.202066] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/12/2020] [Accepted: 04/23/2021] [Indexed: 05/03/2023]
Abstract
Long-term use of zoledronic acid (ZA) increases the risk of medication-related osteonecrosis of the jaw (MRONJ). This may be attributed to ZA-mediated reduction of viable mesenchymal stem cells (MSCs). ZA inhibits protein geranylgeranylation, thus suppressing cell viability and proliferation. Geranylgeraniol (GGOH), which is a naturally found intermediate compound in the mevalonate pathway, has positive effects against ZA. However, precise mechanisms by which GGOH may help preserve stem cell viability against ZA are not fully understood. The objective of this study was to investigate the cytoprotective mechanisms of GGOH against ZA. The results showed that while ZA dramatically decreased the number of viable MSCs, GGOH prevented this negative effect. GGOH-rescued ZA-exposed MSCs formed mineralization comparable to that produced by normal MSCs. Mechanistically, GGOH preserved the number of viable MSCs by its reversal of ZA-mediated Ki67+ MSC number reduction, cell cycle arrest and apoptosis. Moreover, GGOH prevented ZA-suppressed RhoA activity and YAP activation. The results also established the involvement of Rho-dependent YAP and YAP-mediated CDK6 in the cytoprotective ability of GGOH against ZA. In conclusion, GGOH preserves a pool of viable MSCs with osteogenic potency against ZA by rescuing the activity of Rho-dependent YAP activation, suggesting GGOH as a promising agent and YAP as a potential therapeutic target for MRONJ.
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Affiliation(s)
- Weerachai Singhatanadgit
- Faculty of Dentistry, Thammasat University, Pathumthani, 12121, Thailand
- Research Unit in Mineralized Tissue Reconstruction, Thammasat University, Pathumthani, 12121, Thailand
| | - Weerawan Hankamolsiri
- Biofunctional Materials and Devices Research Group, National Metal and Materials Technology Center, Pathumthani 12120, Thailand
| | - Wanida Janvikul
- Biofunctional Materials and Devices Research Group, National Metal and Materials Technology Center, Pathumthani 12120, Thailand
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15
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Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of RhoA/ROCK/MLC Signaling Pathway. J Virol 2021; 95:JVI.00051-21. [PMID: 33692204 PMCID: PMC8139687 DOI: 10.1128/jvi.00051-21] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Tight junctions (TJs) are a major barrier and also an important portal of entry for different pathogens. Porcine sapovirus (PSaV) induces early disruption of the TJ integrity of polarized LLC-PK cells, allowing it to bind to the buried occludin co-receptors hidden beneath the TJs on the basolateral surface. However, the signaling pathways involved in the PSaV-induced TJ dissociation are not yet known. Here, we found that the RhoA/ROCK/MLC signaling pathway was activated in polarized LLC-PK cells during the early infection of PSaV Cowden strain in the presence of bile acid. Specific inhibitors of RhoA, ROCK, and MLC restored PSaV-induced reduction of transepithelial resistance, increase of paracellular flux, intracellular translocation of occludin, and lateral membrane lipid diffusion. Moreover, each inhibitor significantly reduced PSaV replication, as evidenced by a reduction in viral protein synthesis, genome copy number, and progeny viruses. The PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, known to dissociate TJs, were not activated during early PSaV infection. Among the above signaling pathways, the RhoA/ROCK/MLC signaling pathway was only activated by PSaV in the absence of bile acid, and specific inhibitors of this signaling pathway restored early TJ dissociation. Our findings demonstrate that PSaV binding to cell surface receptors activates the RhoA/ROCK/MLC signaling pathway, which in turn disrupts TJ integrity via the contraction of the actomyosin ring. Our study contributes to understanding how PSaV enters the cells and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections.IMPORTANCEPorcine sapovirus (PSaV), one of the most important enteric pathogens, is known to disrupt tight junction (TJ) integrity to expose its buried co-receptor occludin in polarized LLC-PK cells. However, the cellular signaling pathways that facilitate TJ dissociation are not yet completely understood. Here, we demonstrate that early infection of PSaV in polarized LLC-PK cells in either the presence or absence of bile acids activates the RhoA/ROCK/MLC signaling pathway, whose inhibitors reverse the early PSaV infection-induced early dissociation of TJs and reduce PSaV replication. However, early PSaV infection did not activate the PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, which are also known to dissociate TJs. This study provides a better understanding of the mechanism involved in early PSaV infection-induced disruption of TJs, which is important for controlling or preventing PSaV and other calicivirus infections.
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16
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Geoghegan IP, McNamara LM, Hoey DA. Estrogen withdrawal alters cytoskeletal and primary ciliary dynamics resulting in increased Hedgehog and osteoclastogenic paracrine signalling in osteocytes. Sci Rep 2021; 11:9272. [PMID: 33927279 PMCID: PMC8085225 DOI: 10.1038/s41598-021-88633-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2021] [Accepted: 04/12/2021] [Indexed: 01/02/2023] Open
Abstract
Estrogen deficiency during post-menopausal osteoporosis leads to osteoclastogenesis and bone loss. Increased pro-osteoclastogenic signalling (RANKL/OPG) by osteocytes occurs following estrogen withdrawal (EW) and is associated with impaired focal adhesions (FAs) and a disrupted actin cytoskeleton. RANKL production is mediated by Hedgehog signalling in osteocytes, a signalling pathway associated with the primary cilium, and the ciliary structure is tightly coupled to the cytoskeleton. Therefore, the objective of this study was to investigate the role of the cilium and associated signalling in EW-mediated osteoclastogenic signalling in osteocytes. We report that EW leads to an elongation of the cilium and increase in Hedgehog and osteoclastogenic signalling. Significant trends were identified linking cilia elongation with reductions in cell area and % FA area/cell area, indicating that cilia elongation is associated with disruption of FAs and actin contractility. To verify this, we inhibited FA assembly via αvβ3 antagonism and inhibited actin contractility and demonstrated an elongated cilia and increased expression of Hh markers and Rankl expression. Therefore, our results suggest that the EW conditions associated with osteoporosis lead to a disorganisation of αvβ3 integrins and reduced actin contractility, which were associated with an elongation of the cilium, activation of the Hh pathway and osteoclastogenic paracrine signalling.
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Affiliation(s)
- Ivor P Geoghegan
- Mechanobiology and Medical Devices Research Group, Biomedical Engineering, College of Science and Engineering, National University of Ireland, Galway, Ireland.,Centre for Research in Medical Devices (CÚRAM), National University of Ireland, Galway, Ireland
| | - Laoise M McNamara
- Mechanobiology and Medical Devices Research Group, Biomedical Engineering, College of Science and Engineering, National University of Ireland, Galway, Ireland.,Centre for Research in Medical Devices (CÚRAM), National University of Ireland, Galway, Ireland
| | - David A Hoey
- Centre for Research in Medical Devices (CÚRAM), National University of Ireland, Galway, Ireland. .,Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, D02 R590, Ireland. .,Department of Mechanical, Manufacturing, and Biomedical Engineering, School of Engineering, Trinity College Dublin, Dublin, Ireland. .,Advanced Materials and Bioengineering Research Centre, Trinity College Dublin & RCSI, Dublin 2, Ireland.
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17
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Mondrinos MJ, Alisafaei F, Yi AY, Ahmadzadeh H, Lee I, Blundell C, Seo J, Osborn M, Jeon TJ, Kim SM, Shenoy VB, Huh D. Surface-directed engineering of tissue anisotropy in microphysiological models of musculoskeletal tissue. SCIENCE ADVANCES 2021; 7:7/11/eabe9446. [PMID: 33712463 PMCID: PMC7954445 DOI: 10.1126/sciadv.abe9446] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 01/27/2021] [Indexed: 05/11/2023]
Abstract
Here, we present an approach to model and adapt the mechanical regulation of morphogenesis that uses contractile cells as sculptors of engineered tissue anisotropy in vitro. Our method uses heterobifunctional cross-linkers to create mechanical boundary constraints that guide surface-directed sculpting of cell-laden extracellular matrix hydrogel constructs. Using this approach, we engineered linearly aligned tissues with structural and mechanical anisotropy. A multiscale in silico model of the sculpting process was developed to reveal that cell contractility increases as a function of principal stress polarization in anisotropic tissues. We also show that the anisotropic biophysical microenvironment of linearly aligned tissues potentiates soluble factor-mediated tenogenic and myogenic differentiation of mesenchymal stem cells. The application of our method is demonstrated by (i) skeletal muscle arrays to screen therapeutic modulators of acute oxidative injury and (ii) a 3D microphysiological model of lung cancer cachexia to study inflammatory and oxidative muscle injury induced by tumor-derived signals.
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Affiliation(s)
- Mark J Mondrinos
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Farid Alisafaei
- Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Alex Y Yi
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Hossein Ahmadzadeh
- Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Insu Lee
- Department of Mechanical Engineering, Inha University, Incheon, Korea
| | - Cassidy Blundell
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Jeongyun Seo
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Matthew Osborn
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Tae-Joon Jeon
- Department of Biological Engineering, Inha University, Incheon, Korea
| | - Sun Min Kim
- Department of Mechanical Engineering, Inha University, Incheon, Korea
| | - Vivek B Shenoy
- Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
- NSF Science and Technology Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Dongeun Huh
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
- NSF Science and Technology Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104, USA
- Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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18
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Katoh K. Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase. Biomedicines 2021; 9:biomedicines9020135. [PMID: 33535441 PMCID: PMC7912711 DOI: 10.3390/biomedicines9020135] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 01/22/2021] [Accepted: 01/26/2021] [Indexed: 11/20/2022] Open
Abstract
Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.
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Affiliation(s)
- Kazuo Katoh
- Laboratory of Human Anatomy and Cell Biology, Faculty of Health Sciences, Tsukuba University of Technology, Tsukuba-city, Ibaraki 305-8521, Japan
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19
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Saadeldin IM, Tukur HA, Aljumaah RS, Sindi RA. Rocking the Boat: The Decisive Roles of Rho Kinases During Oocyte, Blastocyst, and Stem Cell Development. Front Cell Dev Biol 2021; 8:616762. [PMID: 33505968 PMCID: PMC7829335 DOI: 10.3389/fcell.2020.616762] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2020] [Accepted: 12/07/2020] [Indexed: 01/09/2023] Open
Abstract
The rho-associated coiled-coil-containing proteins (ROCKs or rho kinase) are effectors of the small rho-GTPase rhoA, which acts as a signaling molecule to regulate a variety of cellular processes, including cell proliferation, adhesion, polarity, cytokinesis, and survival. Owing to the multifunctionality of these kinases, an increasing number of studies focus on understanding the pleiotropic effects of the ROCK signaling pathway in the coordination and control of growth (proliferation, initiation, and progression), development (morphology and differentiation), and survival in many cell types. There is growing evidence that ROCKs actively phosphorylate several actin-binding proteins and intermediate filament proteins during oocyte cytokinesis, the preimplantation embryos as well as the stem cell development and differentiation. In this review, we focus on the participation of ROCK proteins in oocyte maturation, blastocyst formation, and stem cell development with a special focus on the selective targeting of ROCK isoforms, ROCK1, and ROCK2. The selective switching of cell fate through ROCK inhibition would provide a novel paradigm for in vitro oocyte maturation, experimental embryology, and clinical applications.
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Affiliation(s)
- Islam M Saadeldin
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.,Department of Comparative Medicine, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
| | - Hammed A Tukur
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
| | - Riyadh S Aljumaah
- Department of Animal Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
| | - Ramya A Sindi
- Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi Arabia
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20
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Cheng Y, Felix B, Othmer HG. The Roles of Signaling in Cytoskeletal Changes, Random Movement, Direction-Sensing and Polarization of Eukaryotic Cells. Cells 2020; 9:E1437. [PMID: 32531876 PMCID: PMC7348768 DOI: 10.3390/cells9061437] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 05/28/2020] [Accepted: 05/29/2020] [Indexed: 12/21/2022] Open
Abstract
Movement of cells and tissues is essential at various stages during the lifetime of an organism, including morphogenesis in early development, in the immune response to pathogens, and during wound-healing and tissue regeneration. Individual cells are able to move in a variety of microenvironments (MEs) (A glossary of the acronyms used herein is given at the end) by suitably adapting both their shape and how they transmit force to the ME, but how cells translate environmental signals into the forces that shape them and enable them to move is poorly understood. While many of the networks involved in signal detection, transduction and movement have been characterized, how intracellular signals control re-building of the cyctoskeleton to enable movement is not understood. In this review we discuss recent advances in our understanding of signal transduction networks related to direction-sensing and movement, and some of the problems that remain to be solved.
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Affiliation(s)
- Yougan Cheng
- Bristol Myers Squibb, Route 206 & Province Line Road, Princeton, NJ 08543, USA;
| | - Bryan Felix
- School of Mathematics, University of Minnesota, Minneapolis, MN 55445, USA;
| | - Hans G. Othmer
- School of Mathematics, University of Minnesota, Minneapolis, MN 55445, USA;
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21
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Zhang J, Alisafaei F, Nikolić M, Nou XA, Kim H, Shenoy VB, Scarcelli G. Nuclear Mechanics within Intact Cells Is Regulated by Cytoskeletal Network and Internal Nanostructures. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2020; 16:e1907688. [PMID: 32243075 PMCID: PMC7799396 DOI: 10.1002/smll.201907688] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 03/05/2020] [Accepted: 03/05/2020] [Indexed: 05/11/2023]
Abstract
The mechanical properties of the cellular nucleus are extensively studied as they play a critical role in important processes, such as cell migration, gene transcription, and stem cell differentiation. While the mechanical properties of the isolated nucleus have been tested, there is a lack of measurements about the mechanical behavior of the nucleus within intact cells and specifically about the interplay of internal nuclear components with the intracellular microenvironment, because current testing methods are based on contact and only allow studying the nucleus after isolation from a cell or disruption of cytoskeleton. Here, all-optical Brillouin microscopy and 3D chemomechanical modeling are used to investigate the regulation of nuclear mechanics in physiological conditions. It is observed that the nuclear modulus can be modulated by epigenetic regulation targeting internal nuclear nanostructures such as lamin A/C and chromatin. It is also found that nuclear modulus is strongly regulated by cytoskeletal behavior through a robust mechanism conserved in different culturing conditions. Given the active role of cytoskeletal modulation in nearly all cell functions, this work will enable to reveal highly relevant mechanisms of nuclear mechanical regulations in physiological and pathological conditions.
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Affiliation(s)
- Jitao Zhang
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Farid Alisafaei
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, PA, 19104, USA
- Center for Engineering Mechanobiology, University of Pennsylvania, PA, 19104, USA
| | - Miloš Nikolić
- Maryland Biophysics Program, University of Maryland, College Park, MD 20742, USA
| | - Xuefei A. Nou
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
| | - Hanyoup Kim
- Canon U.S. Life Sciences, Inc., 9800 Medical Center Drive, Suite C-120, Rockville, MD 20850, USA
| | - Vivek B. Shenoy
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, PA, 19104, USA
- Center for Engineering Mechanobiology, University of Pennsylvania, PA, 19104, USA
| | - Giuliano Scarcelli
- Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA
- Maryland Biophysics Program, University of Maryland, College Park, MD 20742, USA
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Kłopocka W, Korczyński J, Pomorski P. Cytoskeleton and Nucleotide Signaling in Glioma C6 Cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1202:109-128. [PMID: 32034711 DOI: 10.1007/978-3-030-30651-9_6] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
This chapter describes signaling pathways, stimulated by the P2Y2 nucleotide receptor (P2Y2R), that regulate cellular processes dependent on actin cytoskeleton dynamics in glioma C6 cells. P2Y2R coupled with G-proteins, in response to ATP or UTP, regulates the level of iphosphatidylinositol-4,5-bisphosphate (PIP2) which modulates a variety of actin binding proteins and is involved in calcium response and activates Rac1 and RhoA proteins. The RhoA/ROCK signaling pathway plays an important role in contractile force generation needed for the assembly of stress fibers, focal adhesions and for tail retraction during cell migration. Blocking of this pathway by a specific Rho-kinase inhibitor induces changes in F-actin organization and cell shape and decreases the level of phosphorylated myosin II and cofilin. In glioma C6 cells these changes are reversed after UTP stimulation of P2Y2R. Signaling pathways responsible for this compensation are calcium signaling which regulates MLC kinase activation via calmodulin, and the Rac1/PAK/LIMK cascade. Stimulation of the Rac1 mediated pathway via Go proteins needs additional interaction between αvβ5 integrins and P2Y2Rs. Calcium free medium, or growing of the cells in suspension, prevents Gαo activation by P2Y2 receptors. Rac1 activation is necessary for cofilin phosphorylation as well as integrin activation needed for focal complexes formation and stabilization of lamellipodium. Inhibition of positive Rac1 regulation prevents glioma C6 cells from recovery of control cell like morphology.
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Affiliation(s)
- Wanda Kłopocka
- Faculty of Biology and Environmental Sciences, Cardinal Stefan Wyszynski University, Warsaw, Poland.
| | - Jarosław Korczyński
- M. Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland
| | - Paweł Pomorski
- M. Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warsaw, Poland
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Deng JT, Bhaidani S, Sutherland C, MacDonald JA, Walsh MP. Rho-associated kinase and zipper-interacting protein kinase, but not myosin light chain kinase, are involved in the regulation of myosin phosphorylation in serum-stimulated human arterial smooth muscle cells. PLoS One 2019; 14:e0226406. [PMID: 31834925 PMCID: PMC6910671 DOI: 10.1371/journal.pone.0226406] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Accepted: 11/26/2019] [Indexed: 01/09/2023] Open
Abstract
Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30–120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.
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Affiliation(s)
- Jing-Ti Deng
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Sabreena Bhaidani
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Cindy Sutherland
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Justin A. MacDonald
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
| | - Michael P. Walsh
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
- * E-mail:
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24
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Shen Y, Bu L, Li R, Chen Z, Tian F, Ge Q. Expression And Biological Interaction Network Of RHOC For Hepatic Carcinoma With Metastasis In PBMC Samples. Onco Targets Ther 2019; 12:9117-9128. [PMID: 31806997 PMCID: PMC6842290 DOI: 10.2147/ott.s222235] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2019] [Accepted: 10/02/2019] [Indexed: 12/31/2022] Open
Abstract
Objectives Hepatic carcinoma with metastasis remains incurable, and clinical diagnostic methods lacked adequate sensitivity and specificity. Therefore, seeking effectively diagnostic biomarkers is still essential for it. RHOC was reported to be linked to metastasis of hepatic carcinoma. However, almost all of the studies used tissues as detection samples, which was not ideal for clinical course minoring. Therefore, here, it was aimed to use PBMC samples that were not only easily accessible but also minimally invasive to determine the expression and biological interaction network of RHOC for hepatic carcinoma with metastasis. Methods PBMC samples were isolated. Then, RNA-seq was performed to identify the DEGs between hepatic carcinoma with metastasis and hepatic carcinoma with solitary tumor. Subsequently, q-RT-PCR was used to verify the expression level of RHOC. Finally, bioinformatic analysis was used to present the biological interaction network of RHOC for hepatic carcinoma with metastasis in PBMC samples. Results The results of both RNA-seq and q-RT-PCR showed that the expression level of RHOC was significantly higher in the PBMC samples of hepatic carcinoma with metastasis than in those of hepatic carcinoma with solitary tumor. By using variety of bioinformatic analysis platforms, in PBMCs, 18 co-expression genes with RHOC were identified and their interaction network showed that MYL9 and RHOC had the highest edge evidence, and were involved in some cell migration-related pathways. Conclusion Our results indicated that RHOC in PBMCs could be potentially minimally invasive indicators for the diagnosis and clinical course supervision of hepatic carcinoma with metastasis, and its biological interaction network determined based on bioinformatic methods would lay a foundation for further study of the role of RHOC in tumor invasion and metastasis.
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Affiliation(s)
- Yanting Shen
- Department of Science and Education, Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai 201700, People's Republic of China
| | - Lu Bu
- Department of Interventional Radiology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009, People's Republic of China
| | - Rui Li
- State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People's Republic of China
| | - Zhenzhu Chen
- State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People's Republic of China
| | - Fei Tian
- State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People's Republic of China
| | - Qinyu Ge
- State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, People's Republic of China
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25
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Helical structure of actin stress fibers and its possible contribution to inducing their direction-selective disassembly upon cell shortening. Biomech Model Mechanobiol 2019; 19:543-555. [PMID: 31549258 DOI: 10.1007/s10237-019-01228-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Accepted: 09/10/2019] [Indexed: 12/21/2022]
Abstract
Mechanisms of the assembly of actin stress fibers (SFs) have been extensively studied, while those of the disassembly-particularly cell shortening-induced ones-remain unclear. Here, we show that SFs have helical structures composed of multi-subbundles, and they tend to be delaminated upon cell shortening. Specifically, we observed with atomic force microscopy delamination of helical SFs into their subbundles. We physically caught individual SFs using a pair of glass needles to observe rotational deformations during stretching as well as ATP-driven active contraction, suggesting that they deform in a manner reflecting their intrinsic helical structure. A minimal analytical model was then developed based on the Frenet-Serret formulas with force-strain measurement data to suggest that helical SFs can be delaminated into the constituent subbundles upon axial shortening. Given that SFs are large molecular clusters that bear cellular tension but must promptly disassemble upon loss of the tension, the resulting increase in their surface area due to the shortening-induced delamination may facilitate interaction with surrounding molecules to aid subsequent disintegration. Thus, our results suggest a new mechanism of the disassembly that occurs only in the specific SFs exposed to forced shortening.
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26
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Alisafaei F, Jokhun DS, Shivashankar GV, Shenoy VB. Regulation of nuclear architecture, mechanics, and nucleocytoplasmic shuttling of epigenetic factors by cell geometric constraints. Proc Natl Acad Sci U S A 2019; 116:13200-13209. [PMID: 31209017 PMCID: PMC6613080 DOI: 10.1073/pnas.1902035116] [Citation(s) in RCA: 153] [Impact Index Per Article: 25.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Cells sense mechanical signals from their microenvironment and transduce them to the nucleus to regulate gene expression programs. To elucidate the physical mechanisms involved in this regulation, we developed an active 3D chemomechanical model to describe the three-way feedback between the adhesions, the cytoskeleton, and the nucleus. The model shows local tensile stresses generated at the interface of the cell and the extracellular matrix regulate the properties of the nucleus, including nuclear morphology, levels of lamin A,C, and histone deacetylation, as these tensile stresses 1) are transmitted to the nucleus through cytoskeletal physical links and 2) trigger an actomyosin-dependent shuttling of epigenetic factors. We then show how cell geometric constraints affect the local tensile stresses and subsequently the three-way feedback and induce cytoskeleton-mediated alterations in the properties of the nucleus such as nuclear lamina softening, chromatin stiffening, nuclear lamina invaginations, increase in nuclear height, and shrinkage of nuclear volume. We predict a phase diagram that describes how the disruption of cytoskeletal components impacts the feedback and subsequently induce contractility-dependent alterations in the properties of the nucleus. Our simulations show that these changes in contractility levels can be also used as predictors of nucleocytoplasmic shuttling of transcription factors and the level of chromatin condensation. The predictions are experimentally validated by studying the properties of nuclei of fibroblasts on micropatterned substrates with different shapes and areas.
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Affiliation(s)
- Farid Alisafaei
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104
| | | | - G V Shivashankar
- Mechanobiology Institute, National University of Singapore, 117411, Singapore
- Department of Biological Sciences, National University of Singapore, 117411, Singapore
- FIRC Institute for Molecular Oncology (IFOM), 20139 Milan, Italy
| | - Vivek B Shenoy
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104;
- Center for Engineering Mechanobiology, University of Pennsylvania, Philadelphia, PA 19104
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27
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Vestre K, Kjos I, Guadagno NA, Borg Distefano M, Kohler F, Fenaroli F, Bakke O, Progida C. Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity. Cell Mol Life Sci 2019; 76:2593-2614. [PMID: 30830239 PMCID: PMC11105640 DOI: 10.1007/s00018-019-03057-w] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2018] [Revised: 02/08/2019] [Accepted: 02/26/2019] [Indexed: 12/22/2022]
Abstract
Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.
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Affiliation(s)
- Katharina Vestre
- Department of Biosciences, University of Oslo, Oslo, Norway
- Centre for Immune Regulation, University of Oslo, Oslo, Norway
| | - Ingrid Kjos
- Department of Biosciences, University of Oslo, Oslo, Norway
- Centre for Immune Regulation, University of Oslo, Oslo, Norway
| | - Noemi Antonella Guadagno
- Department of Biosciences, University of Oslo, Oslo, Norway
- Centre for Immune Regulation, University of Oslo, Oslo, Norway
| | - Marita Borg Distefano
- Department of Biosciences, University of Oslo, Oslo, Norway
- Centre for Immune Regulation, University of Oslo, Oslo, Norway
| | - Felix Kohler
- Department of Physics, The NJORD Centre, University of Oslo, Oslo, Norway
| | | | - Oddmund Bakke
- Department of Biosciences, University of Oslo, Oslo, Norway
- Centre for Immune Regulation, University of Oslo, Oslo, Norway
| | - Cinzia Progida
- Department of Biosciences, University of Oslo, Oslo, Norway.
- Centre for Immune Regulation, University of Oslo, Oslo, Norway.
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Emerging Roles of the Endoplasmic Reticulum Associated Unfolded Protein Response in Cancer Cell Migration and Invasion. Cancers (Basel) 2019; 11:cancers11050631. [PMID: 31064137 PMCID: PMC6562633 DOI: 10.3390/cancers11050631] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2019] [Revised: 04/29/2019] [Accepted: 05/01/2019] [Indexed: 12/21/2022] Open
Abstract
Endoplasmic reticulum (ER) proteostasis is often altered in tumor cells due to intrinsic (oncogene expression, aneuploidy) and extrinsic (environmental) challenges. ER stress triggers the activation of an adaptive response named the Unfolded Protein Response (UPR), leading to protein translation repression, and to the improvement of ER protein folding and clearance capacity. The UPR is emerging as a key player in malignant transformation and tumor growth, impacting on most hallmarks of cancer. As such, the UPR can influence cancer cells’ migration and invasion properties. In this review, we overview the involvement of the UPR in cancer progression. We discuss its cross-talks with the cell migration and invasion machinery. Specific aspects will be covered including extracellular matrix (ECM) remodeling, modification of cell adhesion, chemo-attraction, epithelial-mesenchymal transition (EMT), modulation of signaling pathways associated with cell mobility, and cytoskeleton remodeling. The therapeutic potential of targeting the UPR to treat cancer will also be considered with specific emphasis in the impact on metastasis and tissue invasion.
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29
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Matsui TS, Deguchi S. Spatially selective myosin regulatory light chain regulation is absent in dedifferentiated vascular smooth muscle cells but is partially induced by fibronectin and Klf4. Am J Physiol Cell Physiol 2019; 316:C509-C521. [DOI: 10.1152/ajpcell.00251.2017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
The phosphorylation state of myosin regulatory light chain (MRLC) is central to the regulation of contractility that impacts cellular homeostasis and fate decisions. Rho-kinase (ROCK) and myosin light chain kinase (MLCK) are major kinases for MRLC documented to selectively regulate MRLC in a subcellular position-specific manner; specifically, MLCK in some nonmuscle cell types works in the cell periphery to promote migration, while ROCK does so at the central region to sustain contractility. However, it remains unclear whether or not the spatially selective regulation of the MRLC kinases is universally present in other cell types, including dedifferentiated vascular smooth muscle cells (SMCs). Here, we demonstrate the absence of the spatial regulation in dedifferentiated SMCs using both cell lines and primary cells. Thus, our work is distinct from previous reports on cells with migratory potential. We also observed that the spatial regulation is partly induced upon fibronectin stimulation and Krüppel-like factor 4 overexpression. To find clues to the mechanism, we reveal how the phosphorylation state of MRLC is determined within dedifferentiated A7r5 SMCs under the enzymatic competition among three major regulators ROCK, MLCK, and MRLC phosphatase (MLCP). We show that ROCK, but not MLCK, predominantly regulates the MRLC phosphorylation in a manner distinct from previous in vitro-based and in silico-based reports. In this ROCK-dominating cellular system, the contractility at physiological conditions was regulated at the level of MRLC diphosphorylation, because its monophosphorylation is already saturated. Thus, the present study provides insights into the molecular basis underlying the absence of spatial MRLC regulation in dedifferentiated SMCs.
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Affiliation(s)
- Tsubasa S. Matsui
- Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Osaka, Japan
| | - Shinji Deguchi
- Division of Bioengineering, Graduate School of Engineering Science, Osaka University, Osaka, Japan
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30
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Nalle SC, Zuo L, Ong MLDM, Singh G, Worthylake AM, Choi W, Manresa MC, Southworth AP, Edelblum KL, Baker GJ, Joseph NE, Savage PA, Turner JR. Graft-versus-host disease propagation depends on increased intestinal epithelial tight junction permeability. J Clin Invest 2019; 129:902-914. [PMID: 30667372 PMCID: PMC6355225 DOI: 10.1172/jci98554] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 11/27/2018] [Indexed: 12/15/2022] Open
Abstract
Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer CD8+ effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression.
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Affiliation(s)
- Sam C. Nalle
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
| | - Li Zuo
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
- Anhui Medical University, Hefei, Anhui, China
| | - Ma. Lora Drizella M. Ong
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Gurminder Singh
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Alicia M. Worthylake
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Wangsun Choi
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Mario Cabrero Manresa
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Anna P. Southworth
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Karen L. Edelblum
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
- Department of Pathology & Laboratory Medicine, Center for Inflammation and Immunity, Rutgers New Jersey Medical School, Cancer Center, Newark, New Jersey, USA
| | - Gregory J. Baker
- Laboratory of Systems Pharmacology, Harvard Medical School, Harvard Program in Therapeutic Science, Boston, Massachusetts, USA
| | - Nora E. Joseph
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
| | - Peter A. Savage
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
| | - Jerrold R. Turner
- Department of Pathology, The University of Chicago, Chicago, Illinois, USA
- Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA
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Kallas-Kivi A, Trei A, Stepanjuk A, Ruisu K, Kask K, Pooga M, Maimets T. The role of integrin β1 in the heterogeneity of human embryonic stem cells culture. Biol Open 2018; 7:7/11/bio034355. [PMID: 30385434 PMCID: PMC6262870 DOI: 10.1242/bio.034355] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023] Open
Abstract
The maintenance of the pluripotency of human embryonic stem (hES) cells requires special conditions for culturing. These conditions include specific growth factors containing media and extracellular matrix (ECM) or an appropriate substrate for adhesion. Interactions between the cells and ECM are mediated by integrins, which interact with the components of ECM in active conformation. This study focused on the characterisation of the role of integrin β1 in the adhesion, migration and differentiation of hES cells. Blocking integrin β1 abolished the adhesion of hES cells, decreasing their survival and pluripotency. This effect was in part rescued by the inhibition of RhoA signalling with Y-27632. The presence of Y-27632 increased the migration of hES cells and supported their differentiation into embryoid bodies. The differences in integrin β1 recycling in the phosphorylation of the myosin light chain and in the localisation of TSC2 were observed between the hES cells growing as a single-cell culture and in a colony. The hES cells at the centre and borders of the colony were found to have differences in their morphology, migration and signalling network activity. We concluded that the availability of integrin β1 was essential for the contraction, migration and differentiation ability of hES cells. Summary: The interaction between integrin β1 and the extracellular matrix differs at the centre of the colony and at the periphery, and is crucial for the survival of embryonic stem cells.
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Affiliation(s)
- Ade Kallas-Kivi
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
| | - Annika Trei
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
| | - Artjom Stepanjuk
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
| | - Katrin Ruisu
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
| | - Keiu Kask
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
| | - Margus Pooga
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.,Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
| | - Toivo Maimets
- Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia
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32
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Ma TJ, Zhang ZW, Lu YL, Zhang YY, Tao DC, Liu YQ, Ma YX. CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cells. Exp Mol Med 2018; 50:1-15. [PMID: 30287810 PMCID: PMC6172197 DOI: 10.1038/s12276-018-0156-4] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2018] [Revised: 06/20/2018] [Accepted: 06/22/2018] [Indexed: 02/05/2023] Open
Abstract
Circadian genes control most of the physiological functions in cancer cells, including cell proliferation, migration, and invasion. The CLOCK and BMAL1 complex plays a central role in circadian rhythms. Previous studies have shown that circadian genes may act as oncogenes or tumor-suppressor genes. In addition, F-actin, regulated by RHOA, has been shown to participate in tumor progression. However, the roles of the CLOCK and BMAL1 genes in the regulation of tumor progression via the RHOA-ROCK-CFL pathway remain largely unclear. Here we first indicate that the rearrangement of F-actin is regulated by CLOCK and BMAL1. We found that CLOCK and BMAL1 can upregulate RHOA expression by inhibiting CUL3-mediated ubiquitination and activate RHOA by reducing the interaction between RHOA and RhoGDI. Consequently, CLOCK and BMAL1 control the expression of the components of the RHOA-ROCK-CFL pathway, which alters the dynamics of F-actin/G-actin turnover and promotes cancer cell proliferation, migration, and invasion. In conclusion, our research proposes a novel insight into the role of CLOCK and BMAL1 in tumor cells.
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Affiliation(s)
- Teng-Jiao Ma
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Zhi-Wei Zhang
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Yi-Lu Lu
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Ying-Ying Zhang
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Da-Chang Tao
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Yun-Qiang Liu
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China
| | - Yong-Xin Ma
- Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital and Collaborative Innovation Center, Sichuan University, 610041, Chengdu, China.
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Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells. Sci Rep 2018; 8:13931. [PMID: 30224682 PMCID: PMC6141481 DOI: 10.1038/s41598-018-32352-y] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2018] [Accepted: 09/06/2018] [Indexed: 02/02/2023] Open
Abstract
Intestinal epithelial tight junctions (TJ) are a major barrier restricting the entry of various harmful factors including pathogens; however, they also represent an important entry portal for pathogens. Although the rotavirus-induced early disruption of TJ integrity and targeting of TJ proteins as coreceptors are well-defined, the precise molecular mechanisms involved remain unknown. In the present study, infection of polarized MDCK cells with the species A rotavirus (RVA) strains human DS-1 and bovine NCDV induced a redistribution of TJ proteins into the cytoplasm, a reversible decrease in transepithelial resistance, and an increase in paracellular permeability. RhoA/ROCK/MLC signaling was identified as activated at an early stage of infection, while inhibition of this pathway prevented the rotavirus-induced early disruption of TJ integrity and alteration of TJ protein distribution. Activation of pMYPT, PKC, or MLCK, which are known to participate in TJ dissociation, was not observed in MDCK cells infected with either rotavirus strain. Our data demonstrated that binding of RVA virions or cogent VP8* proteins to cellular receptors activates RhoA/ROCK/MLC signaling, which alters TJ protein distribution and disrupts TJ integrity via contraction of the perijunctional actomyosin ring, facilitating virion access to coreceptors and entry into cells.
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ROCK inhibition in models of neurodegeneration and its potential for clinical translation. Pharmacol Ther 2018; 189:1-21. [DOI: 10.1016/j.pharmthera.2018.03.008] [Citation(s) in RCA: 99] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Koride S, Loza AJ, Sun SX. Epithelial vertex models with active biochemical regulation of contractility can explain organized collective cell motility. APL Bioeng 2018; 2:031906. [PMID: 31069315 PMCID: PMC6324211 DOI: 10.1063/1.5023410] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2018] [Accepted: 06/14/2018] [Indexed: 01/22/2023] Open
Abstract
Collective motions of groups of cells are observed in many biological settings such as embryo development, tissue formation, and cancer metastasis. To effectively model collective cell movement, it is important to incorporate cell specific features such as cell size, cell shape, and cell mechanics, as well as active behavior of cells such as protrusion and force generation, contractile forces, and active biochemical signaling mechanisms that regulate cell behavior. In this paper, we develop a comprehensive model of collective cell migration in confluent epithelia based on the vertex modeling approach. We develop a method to compute cell-cell viscous friction based on the vertex model and incorporate RhoGTPase regulation of cortical myosin contraction. Global features of collective cell migration are examined by computing the spatial velocity correlation function. As active cell force parameters are varied, we found rich dynamical behavior. Furthermore, we find that cells exhibit nonlinear phenomena such as contractile waves and vortex formation. Together our work highlights the importance of active behavior of cells in generating collective cell movement. The vertex modeling approach is an efficient and versatile approach to rigorously examine cell motion in the epithelium.
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Affiliation(s)
- Sarita Koride
- Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA
| | - Andrew J Loza
- Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA
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Othmer HG. Eukaryotic Cell Dynamics from Crawlers to Swimmers. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE 2018; 9. [PMID: 30854030 DOI: 10.1002/wcms.1376] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Movement requires force transmission to the environment, and motile cells are robustly, though not elegantly, designed nanomachines that often can cope with a variety of environmental conditions by altering the mode of force transmission used. As with humans, the available modes range from momentary attachment to a substrate when crawling, to shape deformations when swimming, and at the cellular level this involves sensing the mechanical properties of the environment and altering the mode appropriately. While many types of cells can adapt their mode of movement to their microenvironment (ME), our understanding of how they detect, transduce and process information from the ME to determine the optimal mode is still rudimentary. The shape and integrity of a cell is determined by its cytoskeleton (CSK), and thus the shape changes that may be required to move involve controlled remodeling of the CSK. Motion in vivo is often in response to extracellular signals, which requires the ability to detect such signals and transduce them into the shape changes and force generation needed for movement. Thus the nanomachine is complex, and while much is known about individual components involved in movement, an integrated understanding of motility in even simple cells such as bacteria is not at hand. In this review we discuss recent advances in our understanding of cell motility and some of the problems remaining to be solved.
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Affiliation(s)
- H G Othmer
- School of Mathematics, University of Minnesota
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Yamamoto T, Ugawa Y, Kawamura M, Yamashiro K, Kochi S, Ideguchi H, Takashiba S. Modulation of microenvironment for controlling the fate of periodontal ligament cells: the role of Rho/ROCK signaling and cytoskeletal dynamics. J Cell Commun Signal 2018; 12:369-378. [PMID: 29086204 PMCID: PMC5842188 DOI: 10.1007/s12079-017-0425-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2017] [Accepted: 10/17/2017] [Indexed: 12/20/2022] Open
Abstract
Cells behave in a variety of ways when they perceive changes in their microenvironment; the behavior of cells is guided by their coordinated interactions with growth factors, niche cells, and extracellular matrix (ECM). Modulation of the microenvironment affects the cell morphology and multiple gene expressions. Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) signaling is one of the key regulators of cytoskeletal dynamics and actively and/or passively determines the cell fate, such as proliferation, migration, differentiation, and apoptosis, by reciprocal communication with the microenvironment. During periodontal wound healing, it is important to recruit the residential stem cells into the defect site for regeneration and homeostasis of the periodontal tissue. Periodontal ligament (PDL) cells contain a heterogeneous fibroblast population, including mesenchymal stem cells, and contribute to the reconstruction of tooth-supporting tissues. Therefore, bio-regeneration of PDL cells has been the ultimate goal of periodontal therapy for decades. Recent stem cell researches have shed light on intrinsic ECM properties, providing paradigm shifts in cell fate determination. This review focuses on the role of ROCK activity and the effects of Y-27632, a specific inhibitor of ROCK, in the modulation of ECM-microenvironment. Further, it presents the current understanding of how Rho/ROCK signaling affects the fate determination of stem cells, especially PDL cells. In addition, we have also discussed in detail the underlying mechanisms behind the reciprocal response to the microenvironment.
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Affiliation(s)
- Tadashi Yamamoto
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Yuki Ugawa
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Mari Kawamura
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Keisuke Yamashiro
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Shinsuke Kochi
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Hidetaka Ideguchi
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan
| | - Shogo Takashiba
- Department of Pathophysiology - Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.
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Li YR, Yang WX. Myosins as fundamental components during tumorigenesis: diverse and indispensable. Oncotarget 2018; 7:46785-46812. [PMID: 27121062 PMCID: PMC5216836 DOI: 10.18632/oncotarget.8800] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2016] [Accepted: 04/10/2016] [Indexed: 12/11/2022] Open
Abstract
Myosin is a kind of actin-based motor protein. As the crucial functions of myosin during tumorigenesis have become increasingly apparent, the profile of myosin in the field of cancer research has also been growing. Eighteen distinct classes of myosins have been discovered in the past twenty years and constitute a diverse superfamily. Various myosins share similar structures. They all convert energy from ATP hydrolysis to exert mechanical stress upon interactions with microfilaments. Ongoing research is increasingly suggesting that at least seven kinds of myosins participate in the formation and development of cancer. Myosins play essential roles in cytokinesis failure, chromosomal and centrosomal amplification, multipolar spindle formation and DNA microsatellite instability. These are all prerequisites of tumor formation. Subsequently, myosins activate various processes of tumor invasion and metastasis development including cell migration, adhesion, protrusion formation, loss of cell polarity and suppression of apoptosis. In this review, we summarize the current understanding of the roles of myosins during tumorigenesis and discuss the factors and mechanisms which may regulate myosins in tumor progression. Furthermore, we put forward a completely new concept of “chromomyosin” to demonstrate the pivotal functions of myosins during karyokinesis and how this acts to optimize the functions of the members of the myosin superfamily.
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Affiliation(s)
- Yan-Ruide Li
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, China
| | - Wan-Xi Yang
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, China
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Sakamoto Y, Buchanan RM, Sanchez-Adams J, Guilak F, Sacks MS. On the Functional Role of Valve Interstitial Cell Stress Fibers: A Continuum Modeling Approach. J Biomech Eng 2017; 139:2595420. [PMID: 28024085 DOI: 10.1115/1.4035557] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2016] [Indexed: 01/20/2023]
Abstract
The function of the heart valve interstitial cells (VICs) is intimately connected to heart valve tissue remodeling and repair, as well as the onset and progression of valvular pathological processes. There is yet only very limited knowledge and extant models for the complex three-dimensional VIC internal stress-bearing structures, the associated cell-level biomechanical behaviors, and how they change under varying activation levels. Importantly, VICs are known to exist and function within the highly dynamic valve tissue environment, including very high physiological loading rates. Yet we have no knowledge on how these factors affect VIC function. To this end, we extended our previous VIC computational continuum mechanics model (Sakamoto, et al., 2016, "On Intrinsic Stress Fiber Contractile Forces in Semilunar Heart Valve Interstitial Cells Using a Continuum Mixture Model," J. Mech. Behav. Biomed. Mater., 54(244-258)). to incorporate realistic stress-fiber geometries, force-length relations (Hill model for active contraction), explicit α-smooth muscle actin (α-SMA) and F-actin expression levels, and strain rate. Novel micro-indentation measurements were then performed using cytochalasin D (CytoD), variable KCl molar concentrations, both alone and with transforming growth factor β1 (TGF-β1) (which emulates certain valvular pathological processes) to explore how α-SMA and F-actin expression levels influenced stress fiber responses under quasi-static and physiological loading rates. Simulation results indicated that both F-actin and α-SMA contributed substantially to stress fiber force generation, with the highest activation state (90 mM KCL + TGF-β1) inducing the largest α-SMA levels and associated force generation. Validation was performed by comparisons to traction force microscopy studies, which showed very good agreement. Interestingly, only in the highest activation state was strain rate sensitivity observed, which was captured successfully in the simulations. These unique findings demonstrated that only VICs with high levels of αSMA expression exhibited significant viscoelastic effects. Implications of this study include greater insight into the functional role of α-SMA and F-actin in VIC stress fiber function, and the potential for strain rate-dependent effects in pathological states where high levels of α-SMA occur, which appear to be unique to the valvular cellular in vivo microenvironment.
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Affiliation(s)
- Yusuke Sakamoto
- Center for Cardiovascular Simulation, Institute for Computational Engineering and Sciences, Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712
| | - Rachel M Buchanan
- Center for Cardiovascular Simulation, Institute for Computational Engineering and Sciences, Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712
| | - Johannah Sanchez-Adams
- Departments of Orthopaedic Surgery, Duke University Medical Center, Durham, NC 27710;Departments of Biomedical Engineering, Duke University Medical Center, Durham, NC 27710
| | - Farshid Guilak
- Departments of Orthopaedic Surgery, Washington University, St. Louis, MO 63110;Departments of Biomedical Engineering, Washington University, St. Louis, MO 63110;Departments of Developmental Biology, Washington University, St. Louis, MO 63110
| | - Michael S Sacks
- W. A. "Tex" Moncrief, Jr. Simulation-Based Engineering Science Chair I Center for Cardiovascular Simulation, Institute for Computational Engineering and Sciences, Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712 e-mail:
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40
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Katoh K. Activation of Rho-kinase and focal adhesion kinase regulates the organization of stress fibers and focal adhesions in the central part of fibroblasts. PeerJ 2017; 5:e4063. [PMID: 29158989 PMCID: PMC5694213 DOI: 10.7717/peerj.4063] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Accepted: 10/29/2017] [Indexed: 12/12/2022] Open
Abstract
Specific regulation and activation of focal adhesion kinase (FAK) are thought to be important for focal adhesion formation, and activation of Rho-kinase has been suggested to play a role in determining the effects of FAK on the formation of stress fibers and focal adhesions. To clarify the role of FAK in stress fiber formation and focal adhesion organization, the author examined the formation of new stress fibers and focal adhesions by activation of Rho-kinase in FAK knockout (FAK–/–) fibroblasts. FAK–/– cells were elliptical in shape, and showed reduced numbers of stress fibers and focal adhesions in the central part of the cells along with large focal adhesions in the peripheral regions. Activation of Rho-kinase in FAK–/– cells transiently increased the actin filaments in the cell center, but these did not form typical thick stress fibers. Moreover, only plaque-like structures as the origins of newly formed focal adhesions were observed in the center of the cell. Furthermore, introduction of an exogenous GFP-labeled FAK gene into FAK–/– cells resulted in increased numbers of stress fibers and focal adhesions in the center of the cells, which showed typical fibroblast morphology. These results indicated that FAK plays an important role in the formation of stress fibers and focal adhesions as well as in regulation of cell shape and morphology with the activation of Rho-kinase.
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Affiliation(s)
- Kazuo Katoh
- Laboratory of Human Anatomy and Cell Biology, Faculty of Health Sciences, Tsukuba University of Technology, Tsukuba-city, Ibaraki, Japan
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41
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Cao J, Wang J, Jackman CP, Cox AH, Trembley MA, Balowski JJ, Cox BD, De Simone A, Dickson AL, Di Talia S, Small EM, Kiehart DP, Bursac N, Poss KD. Tension Creates an Endoreplication Wavefront that Leads Regeneration of Epicardial Tissue. Dev Cell 2017; 42:600-615.e4. [PMID: 28950101 DOI: 10.1016/j.devcel.2017.08.024] [Citation(s) in RCA: 96] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 06/24/2017] [Accepted: 08/29/2017] [Indexed: 01/02/2023]
Abstract
Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.
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Affiliation(s)
- Jingli Cao
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA
| | - Jinhu Wang
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA
| | - Christopher P Jackman
- Regeneration Next, Duke University, Durham, NC 27710, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
| | - Amanda H Cox
- Department of Biology, Duke University, Durham, NC 27708, USA
| | - Michael A Trembley
- Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14624, USA; Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY 14624, USA
| | - Joseph J Balowski
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA
| | - Ben D Cox
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA
| | - Alessandro De Simone
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
| | - Amy L Dickson
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA
| | - Stefano Di Talia
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
| | - Eric M Small
- Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14624, USA; Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY 14624, USA; Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14624, USA
| | | | - Nenad Bursac
- Regeneration Next, Duke University, Durham, NC 27710, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
| | - Kenneth D Poss
- Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA; Regeneration Next, Duke University, Durham, NC 27710, USA.
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42
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Petrov D, Dahan I, Cohen-Kfir E, Ravid S. aPKCζ affects directed cell migration through the regulation of myosin light chain phosphorylation. Cell Adh Migr 2017; 11:347-359. [PMID: 27541056 DOI: 10.1080/19336918.2016.1225631] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Cell motility is an essential cellular process for a variety of biological events. It requires cross-talk between the signaling and the cytoskeletal systems. Despite the recognized importance of aPKCζ for cell motility, there is little understanding of the mechanism by which aPKCζ mediates extracellular signals to the cytoskeleton. In the present study, we report that aPKCζ is required for the cellular organization of acto-non-muscle myosin II (NMII) cytoskeleton, for proper cell adhesion and directed cell migration. We show that aPKCζ mediates EGF-dependent RhoA activation and recruitment to the cell membrane. We also show that aPKCζ mediates EGF-dependent myosin light chain (MRLC) phosphorylation that is carried out by Rho-associated protein kinase (ROCK), and that aPKCζ is required for EGF-dependent phosphorylation and inhibition of the myosin phosphatase targeting subunit (MYPT). Finally, we show that aPKCζ mediates the spatial organization of the acto-NMII cytoskeleton in response to EGF stimulation. Our data suggest that aPKCζ is an essential component regulator of acto-NMII cytoskeleton organization leading to directed cell migration, and is a mediator of the EGF signal to the cytoskeleton.
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Affiliation(s)
- Daria Petrov
- a Department of Biochemistry and Molecular Biology , The Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School , Jerusalem , Israel
| | - Inbal Dahan
- a Department of Biochemistry and Molecular Biology , The Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School , Jerusalem , Israel
| | - Einav Cohen-Kfir
- a Department of Biochemistry and Molecular Biology , The Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School , Jerusalem , Israel
| | - Shoshana Ravid
- a Department of Biochemistry and Molecular Biology , The Institute of Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School , Jerusalem , Israel
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Cao X, Moeendarbary E, Isermann P, Davidson PM, Wang X, Chen MB, Burkart AK, Lammerding J, Kamm RD, Shenoy VB. A Chemomechanical Model for Nuclear Morphology and Stresses during Cell Transendothelial Migration. Biophys J 2017; 111:1541-1552. [PMID: 27705776 DOI: 10.1016/j.bpj.2016.08.011] [Citation(s) in RCA: 91] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2016] [Revised: 08/08/2016] [Accepted: 08/15/2016] [Indexed: 10/20/2022] Open
Abstract
It is now evident that the cell nucleus undergoes dramatic shape changes during important cellular processes such as cell transmigration through extracellular matrix and endothelium. Recent experimental data suggest that during cell transmigration the deformability of the nucleus could be a limiting factor, and the morphological and structural alterations that the nucleus encounters can perturb genomic organization that in turn influences cellular behavior. Despite its importance, a biophysical model that connects the experimentally observed nuclear morphological changes to the underlying biophysical factors during transmigration through small constrictions is still lacking. Here, we developed a universal chemomechanical model that describes nuclear strains and shapes and predicts thresholds for the rupture of the nuclear envelope and for nuclear plastic deformation during transmigration through small constrictions. The model includes actin contraction and cytosolic back pressure that squeeze the nucleus through constrictions and overcome the mechanical resistance from deformation of the nucleus and the constrictions. The nucleus is treated as an elastic shell encompassing a poroelastic material representing the nuclear envelope and inner nucleoplasm, respectively. Tuning the chemomechanical parameters of different components such as cell contractility and nuclear and matrix stiffnesses, our model predicts the lower bounds of constriction size for successful transmigration. Furthermore, treating the chromatin as a plastic material, our model faithfully reproduced the experimentally observed irreversible nuclear deformations after transmigration in lamin-A/C-deficient cells, whereas the wild-type cells show much less plastic deformation. Along with making testable predictions, which are in accord with our experiments and existing literature, our work provides a realistic framework to assess the biophysical modulators of nuclear deformation during cell transmigration.
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Affiliation(s)
- Xuan Cao
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Emad Moeendarbary
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Mechanical Engineering, University College London, London, United Kingdom
| | - Philipp Isermann
- Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York
| | - Patricia M Davidson
- Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York
| | - Xiao Wang
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania
| | - Michelle B Chen
- Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Anya K Burkart
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Jan Lammerding
- Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York; Nancy C. and Peter E. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York
| | - Roger D Kamm
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts
| | - Vivek B Shenoy
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania.
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Raya-Sandino A, Castillo-Kauil A, Domínguez-Calderón A, Alarcón L, Flores-Benitez D, Cuellar-Perez F, López-Bayghen B, Chávez-Munguía B, Vázquez-Prado J, González-Mariscal L. Zonula occludens-2 regulates Rho proteins activity and the development of epithelial cytoarchitecture and barrier function. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2017; 1864:1714-1733. [PMID: 28554775 DOI: 10.1016/j.bbamcr.2017.05.016] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2016] [Revised: 05/18/2017] [Accepted: 05/24/2017] [Indexed: 12/11/2022]
Abstract
Silencing Zonula occludens 2 (ZO-2), a tight junctions (TJ) scaffold protein, in epithelial cells (MDCK ZO-2 KD) triggers: 1) Decreased cell to substratum attachment, accompanied by reduced expression of claudin-7 and integrin β1, and increased vinculin recruitment to focal adhesions and stress fibers formation; 2) Lowered cell-cell aggregation and appearance of wider intercellular spaces; 3) Increased RhoA/ROCK activity, mediated by GEF-HI recruitment to cell borders by cingulin; 4) Increased Cdc42 activity, mitotic spindle disorientation and the appearance of cysts with multiple lumens; 5) Increased Rac and cofilin activity, multiple lamellipodia formation and random cell migration but increased wound closure; 6) Diminished cingulin phosphorylation and disappearance of planar network of microtubules at the TJ region; and 7) Increased transepithelial electrical resistance at steady state, coupled to an increased expression of ZO-1 and claudin-4 and a decreased expression of claudin-2 and paracingulin. Hence, ZO-2 is a crucial regulator of Rho proteins activity and the development of epithelial cytoarchitecture and barrier function.
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Affiliation(s)
- Arturo Raya-Sandino
- Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Alejandro Castillo-Kauil
- Department of Cell Biology, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Alaide Domínguez-Calderón
- Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Lourdes Alarcón
- Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - David Flores-Benitez
- Max-Planck-Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
| | - Francisco Cuellar-Perez
- Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Bruno López-Bayghen
- Department of Toxicology, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Bibiana Chávez-Munguía
- Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - José Vázquez-Prado
- Department of Pharmacology, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico
| | - Lorenza González-Mariscal
- Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), México D.F. 07360, Mexico.
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Multiscale model predicts increasing focal adhesion size with decreasing stiffness in fibrous matrices. Proc Natl Acad Sci U S A 2017; 114:E4549-E4555. [PMID: 28468803 DOI: 10.1073/pnas.1620486114] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
We describe a multiscale model that incorporates force-dependent mechanical plasticity induced by interfiber cross-link breakage and stiffness-dependent cellular contractility to predict focal adhesion (FA) growth and mechanosensing in fibrous extracellular matrices (ECMs). The model predicts that FA size depends on both the stiffness of ECM and the density of ligands available to form adhesions. Although these two quantities are independent in commonly used hydrogels, contractile cells break cross-links in soft fibrous matrices leading to recruitment of fibers, which increases the ligand density in the vicinity of cells. Consequently, although the size of focal adhesions increases with ECM stiffness in nonfibrous and elastic hydrogels, plasticity of fibrous networks leads to a departure from the well-described positive correlation between stiffness and FA size. We predict a phase diagram that describes nonmonotonic behavior of FA in the space spanned by ECM stiffness and recruitment index, which describes the ability of cells to break cross-links and recruit fibers. The predicted decrease in FA size with increasing ECM stiffness is in excellent agreement with recent observations of cell spreading on electrospun fiber networks with tunable cross-link strengths and mechanics. Our model provides a framework to analyze cell mechanosensing in nonlinear and inelastic ECMs.
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Abstract
Rho family GTPase signaling regulates the actin cytoskeleton and is critical for behaviors that range from the cell to tissue-scale. A theme in Rho GTPase biology is that there are many more regulators, such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), than GTPases themselves. Here, we review different, modular cases where GEFs and GAPs function together to elicit precise spatial and temporal control of signaling. We focus on examples from metazoan development, where precise regulation of Rho GTPases is critical for proper tissue form and function.
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Affiliation(s)
- Marlis Denk-Lobnig
- a Department of Biology , Massachusetts Institute of Technology , Cambridge , MA , USA
| | - Adam C Martin
- a Department of Biology , Massachusetts Institute of Technology , Cambridge , MA , USA
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Henson JH, Ditzler CE, Germain A, Irwin PM, Vogt ET, Yang S, Wu X, Shuster CB. The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis. Mol Biol Cell 2017; 28:613-623. [PMID: 28057763 PMCID: PMC5328620 DOI: 10.1091/mbc.e16-06-0466] [Citation(s) in RCA: 70] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2016] [Revised: 12/16/2016] [Accepted: 12/30/2016] [Indexed: 11/22/2022] Open
Abstract
Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.
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Affiliation(s)
- John H Henson
- Department of Biology, Dickinson College, Carlisle, PA 17013
- Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250
| | - Casey E Ditzler
- Department of Biology, Dickinson College, Carlisle, PA 17013
| | - Aphnie Germain
- Department of Biology, Dickinson College, Carlisle, PA 17013
| | - Patrick M Irwin
- Department of Biology, Dickinson College, Carlisle, PA 17013
| | - Eric T Vogt
- Department of Biology, Dickinson College, Carlisle, PA 17013
| | - Shucheng Yang
- Department of Biology, Dickinson College, Carlisle, PA 17013
| | - Xufeng Wu
- National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20824
| | - Charles B Shuster
- Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250
- Department of Biology, New Mexico State University, Las Cruces, NM 88003
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Ahmadzadeh H, Webster MR, Behera R, Jimenez Valencia AM, Wirtz D, Weeraratna AT, Shenoy VB. Modeling the two-way feedback between contractility and matrix realignment reveals a nonlinear mode of cancer cell invasion. Proc Natl Acad Sci U S A 2017; 114:E1617-E1626. [PMID: 28196892 PMCID: PMC5338523 DOI: 10.1073/pnas.1617037114] [Citation(s) in RCA: 123] [Impact Index Per Article: 15.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Cancer cell invasion from primary tumors is mediated by a complex interplay between cellular adhesions, actomyosin-driven contractility, and the physical characteristics of the extracellular matrix (ECM). Here, we incorporate a mechanochemical free-energy-based approach to elucidate how the two-way feedback loop between cell contractility (induced by the activity of chemomechanical interactions such as Ca2+ and Rho signaling pathways) and matrix fiber realignment and strain stiffening enables the cells to polarize and develop contractile forces to break free from the tumor spheroids and invade into the ECM. Interestingly, through this computational model, we are able to identify a critical stiffness that is required by the matrix to break intercellular adhesions and initiate cell invasion. Also, by considering the kinetics of the cell movement, our model predicts a biphasic invasiveness with respect to the stiffness of the matrix. These predictions are validated by analyzing the invasion of melanoma cells in collagen matrices of varying concentration. Our model also predicts a positive correlation between the elongated morphology of the invading cells and the alignment of fibers in the matrix, suggesting that cell polarization is directly proportional to the stiffness and alignment of the matrix. In contrast, cells in nonfibrous matrices are found to be rounded and not polarized, underscoring the key role played by the nonlinear mechanics of fibrous matrices. Importantly, our model shows that mechanical principles mediated by the contractility of the cells and the nonlinearity of the ECM behavior play a crucial role in determining the phenotype of the cell invasion.
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Affiliation(s)
- Hossein Ahmadzadeh
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104
| | - Marie R Webster
- Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104
| | - Reeti Behera
- Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104
| | - Angela M Jimenez Valencia
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218
- Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, MD 21218
- Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, MD 21218
| | - Denis Wirtz
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218
- Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, MD 21218
- Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, MD 21218
- Department of Oncology, The Johns Hopkins School of Medicine, Baltimore, MD 21218
- Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, MD 21218
| | - Ashani T Weeraratna
- Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104
| | - Vivek B Shenoy
- Department of Materials Science and Engineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104;
- Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA 19104
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Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization. Biochim Biophys Acta Gen Subj 2017; 1861:1009-1023. [PMID: 28188861 DOI: 10.1016/j.bbagen.2017.02.005] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Revised: 02/02/2017] [Accepted: 02/06/2017] [Indexed: 12/12/2022]
Abstract
BACKGROUND Members of Rho-associated coiled-coil kinases (ROCKs) are effectors of Rho family of small GTPases. ROCKs have multiple functions that include regulation of cellular contraction and polarity, adhesion, motility, proliferation, apoptosis, differentiation, maturation and remodeling of the extracellular matrix (ECM). SCOPE OF THE REVIEW Here, we focus on the action of RhoA and RhoA effectors, ROCK1 and ROCK2, in cells related to tissue mineralization: mesenchymal stem cells, chondrocytes, preosteoblasts, osteoblasts, osteocytes, lining cells and osteoclasts. MAJOR CONCLUSIONS The activation of the RhoA/ROCK pathway promotes stress fiber formation and reduces chondrocyte and osteogenic differentiations, in contrast to that in mesenchymal stem cells which stimulated the osteogenic and the chondrogenic differentiation. The effects of Rac1 and Cdc42 in promoting chondrocyte hypertrophy and of Rac1, Rac2 and Cdc42 in osteoclast are discussed. In addition, members of the Rho family of GTPases such Rac1, Rac2, Rac3 and Cdc42, acting upstream of ROCK and/or other protein effectors, may compensate the actions of RhoA, affecting directly or indirectly the actions of ROCKs as well as other protein effectors. GENERAL SIGNIFICANCE ROCK activity can trigger cartilage degradation and affect bone formation, therefore these kinases may represent a possible therapeutic target to treat osteoarthritis and osseous diseases. Inhibition of Rho/ROCK activity in chondrocytes prevents cartilage degradation, stimulate mineralization of osteoblasts and facilitate bone formation around implanted metals. Treatment with osteoprotegerin results in a significant decrease in the expression of Rho GTPases, ROCK1 and ROCK2, reducing bone resorption. Inhibition of ROCK signaling increases osteoblast differentiation in a topography-dependent manner.
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Coordinated cell motility is regulated by a combination of LKB1 farnesylation and kinase activity. Sci Rep 2017; 7:40929. [PMID: 28102310 PMCID: PMC5244416 DOI: 10.1038/srep40929] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 12/14/2016] [Indexed: 01/07/2023] Open
Abstract
Cell motility requires the precise coordination of cell polarization, lamellipodia formation, adhesion, and force generation. LKB1 is a multi-functional serine/threonine kinase that associates with actin at the cellular leading edge of motile cells and suppresses FAK. We sought to understand how LKB1 coordinates these multiple events by systematically dissecting LKB1 protein domain function in combination with live cell imaging and computational approaches. We show that LKB1-actin colocalization is dependent upon LKB1 farnesylation leading to RhoA-ROCK-mediated stress fiber formation, but membrane dynamics is reliant on LKB1 kinase activity. We propose that LKB1 kinase activity controls membrane dynamics through FAK since loss of LKB1 kinase activity results in morphologically defective nascent adhesion sites. In contrast, defective farnesylation mislocalizes nascent adhesion sites, suggesting that LKB1 farnesylation serves as a targeting mechanism for properly localizing adhesion sites during cell motility. Together, we propose a model where coordination of LKB1 farnesylation and kinase activity serve as a multi-step mechanism to coordinate cell motility during migration.
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