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Fois MG, Tahmasebi Birgani ZN, López-Iglesias C, Knoops K, van Blitterswijk C, Giselbrecht S, Habibović P, Truckenmüller RK. In vitro vascularization of 3D cell aggregates in microwells with integrated vascular beds. Mater Today Bio 2024; 29:101260. [PMID: 39391792 PMCID: PMC11466645 DOI: 10.1016/j.mtbio.2024.101260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Revised: 08/20/2024] [Accepted: 09/17/2024] [Indexed: 10/12/2024] Open
Abstract
Most human tissues possess vascular networks supplying oxygen and nutrients. Engineering of functional tissue and organ models or equivalents often require the integration of artificial vascular networks. Several approaches, such as organs on chips and three-dimensional (3D) bioprinting, have been pursued to obtain vasculature and vascularized tissues in vitro. This technical feasibility study proposes a new approach for the in vitro vascularization of 3D microtissues. For this, we thermoform arrays of round-bottom microwells into thin non-porous and porous polymer films/membranes and culture vascular beds on them from which endothelial sprouting occurs in a Matrigel-based 3D extra cellular matrix. We present two possible culture configurations for the microwell-integrated vascular beds. In the first configuration, human umbilical vein endothelial cells (HUVECs) grow on and sprout from the inner wall of the non-porous microwells. In the second one, HUVECs grow on the outer surface of the porous microwells and sprout through the pores toward the inside. These approaches are extended to lymphatic endothelial cells. As a proof of concept, we demonstrate the in vitro vascularization of spheroids from human mesenchymal stem cells and MG-63 human osteosarcoma cells. Our results show the potential of this approach to provide the spheroids with an abundant outer vascular network and the indication of an inner vasculature.
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Affiliation(s)
- Maria G. Fois
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
| | - Zeinab N. Tahmasebi Birgani
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
| | - Carmen López-Iglesias
- Microscopy CORE Lab, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands
| | - Kèvin Knoops
- Microscopy CORE Lab, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 50, 6229 ER, Maastricht, the Netherlands
| | - Clemens van Blitterswijk
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
| | - Stefan Giselbrecht
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
| | - Pamela Habibović
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
| | - Roman K. Truckenmüller
- Department of Instructive Biomaterials Engineering, MERLN Institute for Technology-Inspired Regenerative Medicine, Faculty of Health, Medicine and Life Sciences, Maastricht University, Universiteitssingel 40, 6229 ER, Maastricht, the Netherlands
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Monroy-Romero AX, Nieto-Rivera B, Xiao W, Hautefeuille M. Microvascular Engineering for the Development of a Nonembedded Liver Sinusoid with a Lumen: When Endothelial Cells Do Not Lose Their Edge. ACS Biomater Sci Eng 2024; 10:7054-7072. [PMID: 39390649 DOI: 10.1021/acsbiomaterials.4c00939] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/12/2024]
Abstract
Microvascular engineering seeks to exploit known cell-cell and cell-matrix interactions in the context of vasculogenesis to restore homeostasis or disease development of reliable capillary models in vitro. However, current systems generally focus on recapitulating microvessels embedded in thick gels of extracellular matrix, overlooking the significance of discontinuous capillaries, which play a vital role in tissue-blood exchanges particularly in organs like the liver. In this work, we introduce a novel method to stimulate the spontaneous organization of endothelial cells into nonembedded microvessels. By creating an anisotropic micropattern at the edge of a development-like matrix dome using Marangoni flow, we achieved a long, nonrandom orientation of endothelial cells, laying a premise for stable lumenized microvessels. Our findings revealed a distinctive morphogenetic process leading to mature lumenized capillaries, demonstrated with both murine and human immortalized liver sinusoidal endothelial cell lines (LSECs). The progression of cell migration, proliferation, and polarization was clearly guided by the pattern, initiating the formation of a multicellular cord that caused a deformation spanning extensive regions and generated a wave-like folding of the gel, hinged at a laminin-depleted zone, enveloping the cord with gel proteins. This event marked the onset of lumenogenesis, regulated by the gradual apico-basal polarization of the wrapped cells, leading to the maturation of vessel tight junctions, matrix remodeling, and ultimately the formation of a lumen─recapitulating the development of vessels in vivo. Furthermore, we demonstrate that the process strongly relies on the initial gel edge topography, while the geometry of the vessels can be tuned from a curved to a straight structure. We believe that our facile engineering method, guiding an autonomous self-organization of vessels without the need for supporting cells or complex prefabricated scaffolds, holds promise for future integration into microphysiological systems featuring discontinuous, fenestrated capillaries.
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Affiliation(s)
- Ana Ximena Monroy-Romero
- Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, 03100 Mexico, México
- Laboratoire de Biologie du Développement (UMR 7622), Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France
| | - Brenda Nieto-Rivera
- Laboratoire de Biologie du Développement (UMR 7622), Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France
| | - Wenjin Xiao
- Laboratoire de Biologie du Développement (UMR 7622), Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France
| | - Mathieu Hautefeuille
- Laboratoire de Biologie du Développement (UMR 7622), Institut de Biologie Paris Seine, Sorbonne Université, 75005 Paris, France
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Gazsó-Gerhát G, Gombos R, Tóth K, Kaltenecker P, Szikora S, Bíró J, Csapó E, Asztalos Z, Mihály J. FRL and DAAM are required for lateral adhesion of interommatidial cells and patterning of the retinal floor. Development 2023; 150:dev201713. [PMID: 37997920 PMCID: PMC10690107 DOI: 10.1242/dev.201713] [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: 02/17/2023] [Accepted: 10/17/2023] [Indexed: 11/25/2023]
Abstract
Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight with compound eyes. Separation of the ommatidia is ensured by pigment cells that organize into a hexagonal lattice in the Drosophila eye, forming thin walls between the facets. Cell adhesion, mediated by apically and latero-basally located junctional complexes, is crucial for stable attachment of these cells to each other and the basal lamina. Whereas former studies have focused on the formation and remodelling of the cellular connections at the apical region, here, we report a specific alteration of the lateral adhesion of the lattice cells, leaving the apical junctions largely unaffected. We found that DAAM and FRL, two formin-type cytoskeleton regulatory proteins, play redundant roles in lateral adhesion of the interommatidial cells and patterning of the retinal floor. We show that formin-dependent cortical actin assembly is crucial for latero-basal sealing of the ommatidial lattice. We expect that the investigation of these previously unreported eye phenotypes will pave the way toward a better understanding of the three-dimensional aspects of compound eye development.
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Affiliation(s)
- Gabriella Gazsó-Gerhát
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
- Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged H-6726, Hungary
| | - Rita Gombos
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
| | - Krisztina Tóth
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
| | - Péter Kaltenecker
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
| | - Szilárd Szikora
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
| | - Judit Bíró
- Aktogen Hungary Ltd., Szeged H-6726, Hungary
| | - Enikő Csapó
- Aktogen Hungary Ltd., Szeged H-6726, Hungary
| | - Zoltán Asztalos
- Institute of Biochemistry, HUN-REN Biological Research Centre, Szeged H-6726, Hungary
| | - József Mihály
- Institute of Genetics, HUN-REN Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary
- Department of Genetics, University of Szeged, Szeged H-6726, Hungary
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Yadunandanan Nair N, Samuel V, Ramesh L, Marib A, David DT, Sundararaman A. Actin cytoskeleton in angiogenesis. Biol Open 2022; 11:bio058899. [PMID: 36444960 PMCID: PMC9729668 DOI: 10.1242/bio.058899] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/24/2024] Open
Abstract
Actin, one of the most abundant intracellular proteins in mammalian cells, is a critical regulator of cell shape and polarity, migration, cell division, and transcriptional response. Angiogenesis, or the formation of new blood vessels in the body is a well-coordinated multi-step process. Endothelial cells lining the blood vessels acquire several new properties such as front-rear polarity, invasiveness, rapid proliferation and motility during angiogenesis. This is achieved by changes in the regulation of the actin cytoskeleton. Actin remodelling underlies the switch between the quiescent and angiogenic state of the endothelium. Actin forms endothelium-specific structures that support uniquely endothelial functions. Actin regulators at endothelial cell-cell junctions maintain the integrity of the blood-tissue barrier while permitting trans-endothelial leukocyte migration. This review focuses on endothelial actin structures and less-recognised actin-mediated endothelial functions. Readers are referred to other recent reviews for the well-recognised roles of actin in endothelial motility, barrier functions and leukocyte transmigration. Actin generates forces that are transmitted to the extracellular matrix resulting in vascular matrix remodelling. In this review, we attempt to synthesize our current understanding of the roles of actin in vascular morphogenesis. We speculate on the vascular bed specific differences in endothelial actin regulation and its role in the vast heterogeneity in endothelial morphology and function across the various tissues of our body.
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Affiliation(s)
- Nidhi Yadunandanan Nair
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
| | - Victor Samuel
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
| | - Lariza Ramesh
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
| | - Areeba Marib
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
| | - Deena T. David
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
| | - Ananthalakshmy Sundararaman
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India695014
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Ahangar P, Cowin AJ. Reforming the Barrier: The Role of Formins in Wound Repair. Cells 2022; 11:cells11182779. [PMID: 36139355 PMCID: PMC9496773 DOI: 10.3390/cells11182779] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2022] [Revised: 09/02/2022] [Accepted: 09/02/2022] [Indexed: 12/04/2022] Open
Abstract
The restoration of an intact epidermal barrier after wound injury is the culmination of a highly complex and exquisitely regulated physiological process involving multiple cells and tissues, overlapping dynamic events and protein synthesis and regulation. Central to this process is the cytoskeleton, a system of intracellular proteins that are instrumental in regulating important processes involved in wound repair including chemotaxis, cytokinesis, proliferation, migration, and phagocytosis. One highly conserved family of cytoskeletal proteins that are emerging as major regulators of actin and microtubule nucleation, polymerization, and stabilization are the formins. The formin family includes 15 different proteins categorized into seven subfamilies based on three formin homology domains (FH1, FH2, and FH3). The formins themselves are regulated in different ways including autoinhibition, activation, and localization by a range of proteins, including Rho GTPases. Herein, we describe the roles and effects of the formin family of cytoskeletal proteins on the fundamental process of wound healing and highlight recent advances relating to their important functions, mechanisms, and regulation at the molecular and cellular levels.
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Francis CR, Kushner EJ. Trafficking in blood vessel development. Angiogenesis 2022; 25:291-305. [PMID: 35449244 PMCID: PMC9249721 DOI: 10.1007/s10456-022-09838-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Accepted: 04/03/2022] [Indexed: 02/17/2023]
Abstract
Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely studied, area of endothelial cell regulation is vesicular trafficking, also termed sorting. After moving through the Golgi apparatus, proteins are shuttled to organelles, plugged into membranes, recycled, or degraded depending on the internal and extrinsic cues. A snapshot of these protein-sorting systems can be viewed as a trafficking signature that is not only unique to endothelial tissue, but critically important for blood vessel form and function. In this review, we will cover how vesicular trafficking impacts various aspects of angiogenesis, such as sprouting, lumen formation, vessel stabilization, and secretion, emphasizing the role of Rab GTPase family members and their various effectors.
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Affiliation(s)
- Caitlin R Francis
- Department of Biological Sciences, University of Denver, Denver, CO, 80210, USA
| | - Erich J Kushner
- Department of Biological Sciences, University of Denver, Denver, CO, 80210, USA.
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Uemura A, Fukushima Y. Rho GTPases in Retinal Vascular Diseases. Int J Mol Sci 2021; 22:ijms22073684. [PMID: 33916163 PMCID: PMC8036301 DOI: 10.3390/ijms22073684] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2021] [Revised: 03/30/2021] [Accepted: 03/31/2021] [Indexed: 02/06/2023] Open
Abstract
The Rho family of small GTPases (Rho GTPases) act as molecular switches that transduce extrinsic stimuli into cytoskeletal rearrangements. In vascular endothelial cells (ECs), Cdc42, Rac1, and RhoA control cell migration and cell–cell junctions downstream of angiogenic and inflammatory cytokines, thereby regulating vascular formation and permeability. While these Rho GTPases are broadly expressed in various types of cells, RhoJ is enriched in angiogenic ECs. Semaphorin 3E (Sema3E) releases RhoJ from the intracellular domain of PlexinD1, by which RhoJ induces actin depolymerization through competition with Cdc42 for their common effector proteins. RhoJ further mediates the Sema3E-induced association of PlexinD1 with vascular endothelial growth factor receptor (VEGFR) 2 and the activation of p38. Upon stimulation with VEGF-A, RhoJ facilitates the formation of a holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, leading to the prevention of VEGFR2 degradation and the maintenance of intracellular signal transduction. These pleiotropic roles of RhoJ are required for directional EC migration in retinal angiogenesis. This review highlights the latest insights regarding Rho GTPases in the field of vascular biology, as it will be informative to consider their potential as targets for the treatment of aberrant angiogenesis and hyperpermeability in retinal vascular diseases.
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Affiliation(s)
- Akiyoshi Uemura
- Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
- Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Osaka 564-8565, Japan
- Proteo-Science Center, Ehime University, Toon 791-0295, Japan
- Uemura Eye Clinic, Nishinomiya 663-8101, Japan
- Correspondence: ; Tel.: +81-798-61-8000
| | - Yoko Fukushima
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka 563-0871, Japan;
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka 565-0871, Japan
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Wei H, Sundararaman A, Truelsen SLB, Gurevich D, Thastrup J, Mellor H. In Vitro Coculture Assays of Angiogenesis. Methods Mol Biol 2021; 2206:39-46. [PMID: 32754809 DOI: 10.1007/978-1-0716-0916-3_4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.
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Affiliation(s)
- Haoche Wei
- School of Biochemistry, University of Bristol, Bristol, UK
| | | | | | - David Gurevich
- School of Biochemistry, University of Bristol, Bristol, UK
| | | | - Harry Mellor
- School of Biochemistry, University of Bristol, Bristol, UK.
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Faulkner A, Tamiato A, Cathery W, Rampin A, Caravaggi CM, Jover E, Allen S, Mellor H, Hauton D, Heather LC, Spinetti G, Madeddu P. Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus. Diabetologia 2020; 63:2205-2217. [PMID: 32728894 PMCID: PMC7476972 DOI: 10.1007/s00125-020-05230-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/04/2020] [Accepted: 05/22/2020] [Indexed: 12/28/2022]
Abstract
AIMS/HYPOTHESIS Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.
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Affiliation(s)
- Ashton Faulkner
- Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK.
- School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
| | - Anita Tamiato
- Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK
| | - William Cathery
- Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK
| | | | | | - Eva Jover
- Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK
| | - Steve Allen
- Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK
| | - Harry Mellor
- School of Biochemistry, University of Bristol, University Walk, Bristol, BS8 1TD, UK
| | - David Hauton
- Department of Chemistry, University of Oxford, Oxford, UK
| | - Lisa C Heather
- Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford, UK
| | | | - Paolo Madeddu
- Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK.
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Sundararaman A, Mellor H. A functional antagonism between RhoJ and Cdc42 regulates fibronectin remodelling during angiogenesis. Small GTPases 2020; 12:241-245. [PMID: 32857689 PMCID: PMC8205010 DOI: 10.1080/21541248.2020.1809927] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Angiogenesis is the formation of new blood vessels from pre-existing ones. Angiogenesis requires endothelial cells to change shape and polarity, as well as acquire the ability to directionally migrate ‒ processes that are classically regulated by the Rho family of GTPases. RhoJ (previously TCL) is an endothelium enriched Rho GTPase with a 78% amino acid similarity to the ubiquitously expressed Cdc42. In our recent publication, we demonstrate that α5β1 integrin co-traffics with RhoJ. RhoJ specifically represses the internalization of the active α5β1 conformer, leading to a reduced ability of endothelial cells to form fibronectin fibrils. Surprisingly, this function of RhoJ is in opposition to the role of Cdc42, a known driver of fibrillogenesis. Intriguingly, we discovered that the competition for limiting amounts of the shared effector, PAK3, could explain the ability of these two Rho GTPases to regulate fibrillogenesis in opposing directions. Consequently, RhoJ null mice show excessive fibronectin deposition around retinal vessels, possibly due to the unopposed action of Cdc42. Our work suggests that the functional antagonism between RhoJ and Cdc42 could restrict fibronectin remodelling to sites of active angiogenesis to form a provisional matrix for vessel growth. One correlate of our findings is that RhoJ dependent repression of fibronectin remodelling could be atheroprotective in quiescent vessels.
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Affiliation(s)
- Ananthalakshmy Sundararaman
- Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, Kerala, India
| | - Harry Mellor
- School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol, UK
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Sundararaman A, Fukushima Y, Norman JC, Uemura A, Mellor H. RhoJ Regulates α5β1 Integrin Trafficking to Control Fibronectin Remodeling during Angiogenesis. Curr Biol 2020; 30:2146-2155.e5. [PMID: 32302585 DOI: 10.1016/j.cub.2020.03.042] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Revised: 02/03/2020] [Accepted: 03/17/2020] [Indexed: 01/24/2023]
Abstract
Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins-RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3, 4]. RhoJ is required for angiogenesis [5, 6] and has multiple roles in this process [7, 8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5β1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5β1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.
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Affiliation(s)
| | - Yoko Fukushima
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Jim C Norman
- CRUK Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, UK
| | - Akiyoshi Uemura
- Department of Retinal Vascular Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
| | - Harry Mellor
- School of Biochemistry, Biomedical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
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Valdembri D, Serini G. Angiogenesis: The Importance of RHOJ-Mediated Trafficking of Active Integrins. Curr Biol 2020; 30:R652-R654. [PMID: 32516616 DOI: 10.1016/j.cub.2020.04.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
In endothelial cells, trafficking of active α5β1 integrins and polarized fibronectin secretion are important for vascular morphogenesis. A new study unveils how the endothelial small GTPase RHOJ, by repressing trafficking of active α5β1 integrins, controls fibronectin polymerization and in vivo angiogenesis.
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Affiliation(s)
- Donatella Valdembri
- Candiolo Cancer Institute - FPO, IRCCS, 10060 Candiolo (TO), Italy; Department of Oncology, University of Torino School of Medicine, 10060 Candiolo (TO), Italy.
| | - Guido Serini
- Candiolo Cancer Institute - FPO, IRCCS, 10060 Candiolo (TO), Italy; Department of Oncology, University of Torino School of Medicine, 10060 Candiolo (TO), Italy.
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Fukushima Y, Nishiyama K, Kataoka H, Fruttiger M, Fukuhara S, Nishida K, Mochizuki N, Kurihara H, Nishikawa SI, Uemura A. RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells. EMBO J 2020; 39:e102930. [PMID: 32347571 DOI: 10.15252/embj.2019102930] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Revised: 03/30/2020] [Accepted: 04/03/2020] [Indexed: 12/19/2022] Open
Abstract
During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.
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Affiliation(s)
- Yoko Fukushima
- Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan.,Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Koichi Nishiyama
- International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Hiroshi Kataoka
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
| | - Marcus Fruttiger
- UCL Institute of Ophthalmology, University College London, London, UK
| | - Shigetomo Fukuhara
- Department of Molecular Pathophysiology, Institute for Advanced Medical Sciences, Nippon Medical School, Kawasaki, Japan
| | - Kohji Nishida
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Naoki Mochizuki
- Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan
| | - Hiroki Kurihara
- Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Shin-Ichi Nishikawa
- Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan
| | - Akiyoshi Uemura
- Division of Vascular Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe, Japan.,Department of Retinal Vascular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
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14
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Wei H, Sundararaman A, Dickson E, Rennie-Campbell L, Cross E, Heesom KJ, Mellor H. Characterization of the polarized endothelial secretome. FASEB J 2019; 33:12277-12287. [PMID: 31431053 DOI: 10.1096/fj.201900262r] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.
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Affiliation(s)
- Haoche Wei
- Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, Centre for Growth, Metabolism, and Aging, College of Life Sciences, Sichuan University, Chengdu, China.,School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Ananthalakshmy Sundararaman
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Emily Dickson
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Lewis Rennie-Campbell
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Eloise Cross
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Kate J Heesom
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
| | - Harry Mellor
- School of Biochemistry, Biomedical Sciences Building, Faculty of Life Sciences, University of Bristol, Bristol, United Kingdom
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15
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Barbera S, Nardi F, Elia I, Realini G, Lugano R, Santucci A, Tosi GM, Dimberg A, Galvagni F, Orlandini M. The small GTPase Rab5c is a key regulator of trafficking of the CD93/Multimerin-2/β1 integrin complex in endothelial cell adhesion and migration. Cell Commun Signal 2019; 17:55. [PMID: 31138217 PMCID: PMC6537425 DOI: 10.1186/s12964-019-0375-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Accepted: 05/20/2019] [Indexed: 02/06/2023] Open
Abstract
Background In the endothelium, the single-pass membrane protein CD93, through its interaction with the extracellular matrix protein Multimerin-2, activates signaling pathways that are critical for vascular development and angiogenesis. Trafficking of adhesion molecules through endosomal compartments modulates their signaling output. However, the mechanistic basis coordinating CD93 recycling and its implications for endothelial cell (EC) function remain elusive. Methods Human umbilical vein ECs (HUVECs) and human dermal blood ECs (HDBEC) were used in this study. Fluorescence confocal microscopy was employed to follow CD93 retrieval, recycling, and protein colocalization in spreading cells. To better define CD93 trafficking, drug treatments and transfected chimeric wild type and mutant CD93 proteins were used. The scratch assay was used to evaluate cell migration. Gene silencing strategies, flow citometry, and quantification of migratory capability were used to determine the role of Rab5c during CD93 recycling to the cell surface. Results Here, we identify the recycling pathway of CD93 following EC adhesion and migration. We show that the cytoplasmic domain of CD93, by its interaction with Moesin and F-actin, is instrumental for CD93 retrieval in adhering and migrating cells and that aberrant endosomal trafficking of CD93 prevents its localization at the leading edge of migration. Moreover, the small GTPase Rab5c turns out to be a key component of the molecular machinery that is able to drive CD93 recycling to the EC surface. Finally, in the Rab5c endosomal compartment CD93 forms a complex with Multimerin-2 and active β1 integrin, which is recycled back to the basolaterally-polarized cell surface by clathrin-independent endocytosis. Conclusions Our findings, focusing on the pro-angiogenic receptor CD93, unveil the mechanisms of its polarized trafficking during EC adhesion and migration, opening novel therapeutic opportunities for angiogenic diseases. Electronic supplementary material The online version of this article (10.1186/s12964-019-0375-x) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Stefano Barbera
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Federica Nardi
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Ines Elia
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Giulia Realini
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Roberta Lugano
- Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-751 85, Uppsala, Sweden
| | - Annalisa Santucci
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy
| | - Gian Marco Tosi
- Department of Medicine, Surgery and Neuroscience, Ophthalmology Unit, University of Siena, Policlinico "Le Scotte", Viale Bracci, 53100, Siena, Italy
| | - Anna Dimberg
- Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, SE-751 85, Uppsala, Sweden
| | - Federico Galvagni
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy.
| | - Maurizio Orlandini
- Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2, 53100, Siena, Italy.
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16
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Liu J, Chen S, Chen Y, Geng N, Feng C. High expression of FMNL3 associates with cancer cell migration, invasion, and unfavorable prognosis in tongue squamous cell carcinoma. J Oral Pathol Med 2019; 48:459-467. [PMID: 30955218 DOI: 10.1111/jop.12857] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 02/22/2019] [Accepted: 03/17/2019] [Indexed: 01/15/2023]
Affiliation(s)
- Jiameng Liu
- Department of Oral and Maxillofacial Surgery The First Affiliated Hospital Sun Yat‐sen University Guangzhou China
| | - Shan Chen
- Department of Oral and Maxillofacial Surgery The First Affiliated Hospital Sun Yat‐sen University Guangzhou China
| | - Yang Chen
- Department of Oral and Maxillofacial Surgery The First Affiliated Hospital Sun Yat‐sen University Guangzhou China
| | - Ningbo Geng
- Department of Oral and Maxillofacial Surgery The First Affiliated Hospital Sun Yat‐sen University Guangzhou China
| | - Chongjin Feng
- Department of Oral and Maxillofacial Surgery The First Affiliated Hospital Sun Yat‐sen University Guangzhou China
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17
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Wong BS, Shea DJ, Mistriotis P, Tuntithavornwat S, Law RA, Bieber JM, Zheng L, Konstantopoulos K. A Direct Podocalyxin-Dynamin-2 Interaction Regulates Cytoskeletal Dynamics to Promote Migration and Metastasis in Pancreatic Cancer Cells. Cancer Res 2019; 79:2878-2891. [PMID: 30975647 DOI: 10.1158/0008-5472.can-18-3369] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2018] [Revised: 02/18/2019] [Accepted: 04/04/2019] [Indexed: 02/07/2023]
Abstract
The sialoglycoprotein podocalyxin is absent in normal pancreas but is overexpressed in pancreatic cancer and is associated with poor clinical outcome. Here, we investigate the role of podocalyxin in migration and metastasis of pancreatic adenocarcinomas using SW1990 and Pa03c as cell models. Although ezrin is regarded as a cytoplasmic binding partner of podocalyxin that regulates actin polymerization via Rac1 or RhoA, we did not detect podocalyxin-ezrin association in pancreatic cancer cells. Moreover, depletion of podocalyxin did not alter actin dynamics or modulate Rac1 and RhoA activities in pancreatic cancer cells. Using mass spectrometry, bioinformatics analysis, coimmunoprecipitation, and pull-down assays, we discovered a novel, direct binding interaction between the cytoplasmic tail of podocalyxin and the large GTPase dynamin-2 at its GTPase, middle, and pleckstrin homology domains. This podocalyxin-dynamin-2 interaction regulated microtubule growth rate, which in turn modulated focal adhesion dynamics and ultimately promoted efficient pancreatic cancer cell migration via microtubule- and Src-dependent pathways. Depletion of podocalyxin in a hemispleen mouse model of pancreatic cancer diminished liver metastasis without altering primary tumor size. Collectively, these findings reveal a novel mechanism by which podocalyxin facilitates pancreatic cancer cell migration and metastasis. SIGNIFICANCE: These findings reveal that a novel interaction between podocalyxin and dynamin-2 promotes migration and metastasis of pancreatic cancer cells by regulating microtubule and focal adhesion dynamics.
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Affiliation(s)
- Bin Sheng Wong
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland.,Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland
| | - Daniel J Shea
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland
| | - Panagiotis Mistriotis
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland.,Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland
| | - Soontorn Tuntithavornwat
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland
| | - Robert A Law
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland
| | - Jake M Bieber
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland
| | - Lei Zheng
- Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Konstantinos Konstantopoulos
- Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland. .,Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland.,Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland.,Johns Hopkins Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, Maryland.,Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland
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18
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Multiple roles of the actin and microtubule-regulating formins in the developing brain. Neurosci Res 2019; 138:59-69. [DOI: 10.1016/j.neures.2018.09.008] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Revised: 08/22/2018] [Accepted: 08/23/2018] [Indexed: 01/08/2023]
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19
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Zeng YF, Xiao YS, Liu Y, Luo XJ, Wen LD, Liu Q, Chen M. Formin-like 3 regulates RhoC/FAK pathway and actin assembly to promote cell invasion in colorectal carcinoma. World J Gastroenterol 2018; 24:3884-3897. [PMID: 30228782 PMCID: PMC6141330 DOI: 10.3748/wjg.v24.i34.3884] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2018] [Revised: 06/16/2018] [Accepted: 06/27/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To clarify the underlying mechanism of formin-like 3 (FMNL3) in the promotion of colorectal carcinoma (CRC) cell invasion.
METHODS The in vitro biological function analyses of FMNL3 were performed by gain- and loss-of function approaches. Changes in the F-actin cytoskeleton were detected by the technologies of phalloidin-TRITC labeling and confocal microscopy. The signaling pathway mediated by FMNL3 was explored by western blot, gelatin zymograph assay, co-immunoprecipitation (co-IP), immunofluorescence co-localization, and glutathione S-transferase (GST) pull-down assay.
RESULTS The in vitro experimental results showed that FMNL3 significantly promoted the proliferation, invasion, and migration of CRC cells (P < 0.05 and P < 0.01). Moreover, FMNL3 regulated the remodeling of actin-based protrusions such as filopodia and lamellipodia in a RhoC-dependent manner. The western blot and gelatin zymograph assay results indicated that FMNL3 was involved in the RhoC/ focal adhesion kinase (FAK) pathway and acted as an effector of RhoC to activate the downstream signaling of p-FAK as well as p-MAPK and p-AKT. This resulted in the increased expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), and the subsequent promotion of CRC cell invasion. The results of TAE226, U0126 or Ly294002 treatment confirmed an essential role of FMNL3 in activation of the RhoC/FAK pathway and the subsequent promotion of CRC invasion. Co-IP, co-localization and GST pull-down assays showed the direct interaction of FMNL3 with RhoC in vivo and in vitro.
CONCLUSION FMNL3 regulates the RhoC/FAK signaling pathway and RhoC-dependent remodeling of actin-based protrusions to promote CRC invasion.
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Affiliation(s)
- Yuan-Feng Zeng
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Yi-Sheng Xiao
- Teaching and Researching Section of Morphology, College of Basic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi Province, China
| | - Yong Liu
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Xiao-Jiang Luo
- Department of General Surgery, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Li-Dan Wen
- Clinical Medical Sciences Institute, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Qian Liu
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
| | - Min Chen
- Department of Pathology, Jiangxi Provincial People’s Hospital, Nanchang 330006, Jiangxi Province, China
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20
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Szymborska A, Gerhardt H. Hold Me, but Not Too Tight-Endothelial Cell-Cell Junctions in Angiogenesis. Cold Spring Harb Perspect Biol 2018; 10:cshperspect.a029223. [PMID: 28851748 DOI: 10.1101/cshperspect.a029223] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Endothelial cell-cell junctions must perform seemingly incompatible tasks during vascular development-providing stable connections that prevent leakage, while allowing dynamic cellular rearrangements during sprouting, anastomosis, lumen formation, and functional remodeling of the vascular network. This review aims to highlight recent insights into the molecular mechanisms governing endothelial cell-cell adhesion in the context of vascular development.
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Affiliation(s)
- Anna Szymborska
- Integrative Vascular Biology Laboratory, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Berlin
| | - Holger Gerhardt
- Integrative Vascular Biology Laboratory, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.,Vascular Patterning Laboratory, Center for Cancer Biology, VIB, Department of Oncology, KU Leuven, 3000 Leuven, Belgium.,DZHK (German Centre for Cardiovascular Research), partner site Berlin.,Berlin Institute of Health (BIH), 10178 Berlin, Germany
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21
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Heissler SM, Chinthalapudi K, Sellers JR. Kinetic signatures of myosin-5B, the motor involved in microvillus inclusion disease. J Biol Chem 2017; 292:18372-18385. [PMID: 28882893 DOI: 10.1074/jbc.m117.801456] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 08/29/2017] [Indexed: 11/06/2022] Open
Abstract
Myosin-5B is a ubiquitous molecular motor that transports cargo vesicles of the endomembrane system in intracellular recycling pathways. Myosin-5B malfunction causes the congenital enteropathy microvillus inclusion disease, underlining its importance in cellular homeostasis. Here we describe the interaction of myosin-5B with F-actin, nucleotides, and the pyrazolopyrimidine compound myoVin-1. We show that single-headed myosin-5B is an intermediate duty ratio motor with a kinetic ATPase cycle that is rate-limited by the release of phosphate. The presence of a second head generates strain and gating in the myosin-5B dimer that alters the kinetic signature by reducing the actin-activated ADP release rate to become rate-limiting. This kinetic transition into a high-duty ratio motor is a prerequisite for the proposed transport function of myosin-5B in cellular recycling pathways. Moreover, we show that the small molecule compound myoVin-1 inhibits the enzymatic and functional activity of myosin-5B in vitro Partial inhibition of the actin-activated steady-state ATPase activity and sliding velocity suggests that caution should be used when probing the effect of myoVin-1 on myosin-5-dependent transport processes in cells.
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Affiliation(s)
- Sarah M Heissler
- From the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8015 and
| | - Krishna Chinthalapudi
- the Cell Adhesion Laboratory, Department of Integrative Structural and Computational Biology, Scripps Research Institute, Jupiter, Florida 33458
| | - James R Sellers
- From the Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8015 and
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22
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Lim H, Yu C, Jou T. Galectin‐8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. FASEB J 2017; 31:4917-4927. [DOI: 10.1096/fj.201601386r] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2016] [Accepted: 07/10/2017] [Indexed: 01/17/2023]
Affiliation(s)
- HooiCheng Lim
- Graduate Institute of Molecular MedicineNational Taiwan University Taipei Taiwan
| | - Chun‐Ying Yu
- Graduate Institute of Molecular MedicineNational Taiwan University Taipei Taiwan
- Graduate Institute of Clinical MedicineNational Taiwan University Taipei Taiwan
| | - Tzuu‐Shuh Jou
- Institute of Cellular and Organismic BiologyAcademia Sinica Taipei Taiwan
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23
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The Role of RhoJ in Endothelial Cell Biology and Tumor Pathology. BIOMED RESEARCH INTERNATIONAL 2016; 2016:6386412. [PMID: 27556037 PMCID: PMC4983353 DOI: 10.1155/2016/6386412] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/11/2016] [Revised: 05/24/2016] [Accepted: 06/15/2016] [Indexed: 02/04/2023]
Abstract
Background. RhoJ, an endothelially expressed member of Cdc42 (cell division cycle 42) subfamily of Rho GTPase, plays an important role in endocytic pathway, adipocyte differentiation, endothelial motility, tube formation, and focal adhesion. RhoJ is a selective and effective therapeutic target in tumor tissues or retinopathy. Methods. A systematic review was related to “small Rho GTPase” or “RhoJ” with “endothelial motility, tube formation and focal adhesion” and “tumor therapy”. This led to many cross-references involving RhoJ and these data have been incorporated into the following study. Results. We have grouped the role of RhoJ according to three main effects: RhoJ regulates endocytic pathway and adipocyte differentiation in early studies, and RhoJ shows an important role in endothelial cell biology; furthermore, RhoJ blockade serves as a target in tumor vasculature and enhances the effects of anticancer drug. Conclusions. More research is necessary to understand the role of RhoJ in many aspects, on the basis of current knowledge of the role of RhoJ blockade in tumor vessels, there are opportunities for the therapy of tumor, and RhoJ is expressed outside tumour vasculature and is involved in wound healing. Taking advantage of the opportunities could result in a development in tumor therapy.
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24
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Román-Fernández A, Bryant DM. Complex Polarity: Building Multicellular Tissues Through Apical Membrane Traffic. Traffic 2016; 17:1244-1261. [PMID: 27281121 DOI: 10.1111/tra.12417] [Citation(s) in RCA: 58] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Revised: 06/06/2016] [Accepted: 06/06/2016] [Indexed: 12/20/2022]
Abstract
The formation of distinct subdomains of the cell surface is crucial for multicellular organism development. The most striking example of this is apical-basal polarization. What is much less appreciated is that underpinning an asymmetric cell surface is an equally dramatic intracellular endosome rearrangement. Here, we review the interplay between classical cell polarity proteins and membrane trafficking pathways, and discuss how this marriage gives rise to cell polarization. We focus on those mechanisms that regulate apical polarization, as this is providing a number of insights into how membrane traffic and polarity are regulated at the tissue level.
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Affiliation(s)
- Alvaro Román-Fernández
- Cancer Research UK Beatson Institute, Switchback Road, Glasgow, G61 1BD, UK.,Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - David M Bryant
- Cancer Research UK Beatson Institute, Switchback Road, Glasgow, G61 1BD, UK.,Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
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25
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Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila. Genetics 2016; 202:1135-51. [PMID: 26801180 DOI: 10.1534/genetics.115.181438] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2015] [Accepted: 01/18/2016] [Indexed: 01/14/2023] Open
Abstract
The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.
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26
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Formins at the Junction. Trends Biochem Sci 2015; 41:148-159. [PMID: 26732401 DOI: 10.1016/j.tibs.2015.12.002] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Revised: 12/01/2015] [Accepted: 12/04/2015] [Indexed: 12/21/2022]
Abstract
The actin cytoskeleton and adhesion junctions are physically and functionally coupled at the cell-cell interface between epithelial cells. The actin regulatory complex Arp2/3 has an established role in the turnover of junctional actin; however, the role of formins, the largest group of actin regulators, is less clear. Formins dynamically shape the actin cytoskeleton and have various functions within cells. In this review we describe recent progress on how formins regulate actin dynamics at cell-cell contacts and highlight formin functions during polarized protein traffic necessary for epithelialization.
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