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1
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Abstract
The centrosome, consisting of centrioles and the associated pericentriolar material, is the main microtubule-organizing centre (MTOC) in animal cells. During most of interphase, the two centrosomes of a cell are joined together by centrosome cohesion into one MTOC. The most dominant element of centrosome cohesion is the centrosome linker, an interdigitating, fibrous network formed by the protein C-Nap1 anchoring a number of coiled-coil proteins including rootletin to the proximal end of centrioles. Alternatively, centrosomes can be kept together by the action of the minus end directed kinesin motor protein KIFC3 that works on interdigitating microtubules organized by both centrosomes and probably by the actin network. Although cells connect the two interphase centrosomes by several mechanisms into one MTOC, the general importance of centrosome cohesion, particularly for an organism, is still largely unclear. In this article, we review the functions of the centrosome linker and discuss how centrosome cohesion defects can lead to diseases.
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Affiliation(s)
- Hairuo Dang
- Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum-ZMBH Allianz, and,Heidelberg Biosciences International Graduate School (HBIGS), Universität Heidelberg, Heidelberg 69120, Germany
| | - Elmar Schiebel
- Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum-ZMBH Allianz, and
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2
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Hao WW, Xu F. KIFC3 promotes proliferation, migration and invasion of esophageal squamous cell carcinoma cells by activating EMT and β-catenin signaling. World J Gastrointest Oncol 2022; 14:1239-1251. [PMID: 36051093 PMCID: PMC9305573 DOI: 10.4251/wjgo.v14.i7.1239] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 01/26/2022] [Accepted: 03/27/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. A total of 45 kinesin superfamily proteins (KIFs) have been identified in humans, among which several family members have demonstrated varied functions in tumor pathobiology via different mechanisms, including regulation of cell cycle progression and metastasis. KIFC3 has microtubule motor activity and is involved in cancer cell invasion and migration, as well as survival. However, the role of KIFC3 in ESCC is still unknown.
AIM To evaluate the role of KIFC3 in ESCC and the underlying mechanisms.
METHODS Expression of KIFC3 was evaluated in ESCC tissues and adjacent normal esophageal tissues. The prognostic value of KIFC3 was analyzed using Kaplan–Meier Plotter. Colony formation, EdU assays, cell cycle analysis, Transwell assay, immunofluorescence, and western blotting were performed in ESCC cell lines after transfection with pLVX-Puro-KIFC3-shRNA- and pLVX-Puro-KIFC3-expressing lentiviruses. A xenograft tumor model in nude mice was used to evaluate the role of KIFC3 in tumorigenesis. Inhibitor of β-catenin, XAV-939, was used to clarify the mechanism of KIFC3 in ESCC. To analyze the differences between groups, t test and nonparametric tests were used. P < 0.05 was considered statistically significant.
RESULTS Immunohistochemical staining indicated that KIFC3 was upregulated in ESCC tissues compared with adjacent normal tissues. Kaplan–Meier Plotter revealed that overexpressed KIFC3 was associated with poor prognosis in ESCC patients. Colony formation and EdU assay showed that KIFC3 overexpression promoted cell proliferation, while KIFC3 knockdown inhibited cell proliferation in ESCC cell lines. In addition, cell cycle analysis showed that KIFC3 overexpression promoted cell cycle progression. KIFC3 knockdown suppressed ESCC tumorigenesis in vivo. Transwell assay and western blotting revealed that KIFC3 overexpression promoted cell migration and invasion, as well as epithelial–mesenchymal transition (EMT), while KIFC3 knockdown showed the opposite results. Mechanistically, KIFC3 overexpression promoted β-catenin signaling in KYSE450 cells; however, the role of KIFC3 was abolished by XAV-939, the inhibitor of β-catenin signaling.
CONCLUSION KIFC3 was overexpressed in ESCC and was associated with poor prognosis. Furthermore, KIFC3 promoted proliferation, migration and invasion of ESCC via β-catenin signaling and EMT.
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Affiliation(s)
- Wei-Wei Hao
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Feng Xu
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
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3
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Yao M, Qu H, Han Y, Cheng CY, Xiao X. Kinesins in Mammalian Spermatogenesis and Germ Cell Transport. Front Cell Dev Biol 2022; 10:837542. [PMID: 35547823 PMCID: PMC9083010 DOI: 10.3389/fcell.2022.837542] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Accepted: 03/25/2022] [Indexed: 11/13/2022] Open
Abstract
In mammalian testes, the apical cytoplasm of each Sertoli cell holds up to several dozens of germ cells, especially spermatids that are transported up and down the seminiferous epithelium. The blood-testis barrier (BTB) established by neighboring Sertoli cells in the basal compartment restructures on a regular basis to allow preleptotene/leptotene spermatocytes to pass through. The timely transfer of germ cells and other cellular organelles such as residual bodies, phagosomes, and lysosomes across the epithelium to facilitate spermatogenesis is important and requires the microtubule-based cytoskeleton in Sertoli cells. Kinesins, a superfamily of the microtubule-dependent motor proteins, are abundantly and preferentially expressed in the testis, but their functions are poorly understood. This review summarizes recent findings on kinesins in mammalian spermatogenesis, highlighting their potential role in germ cell traversing through the BTB and the remodeling of Sertoli cell-spermatid junctions to advance spermatid transport. The possibility of kinesins acting as a mediator and/or synchronizer for cell cycle progression, germ cell transit, and junctional rearrangement and turnover is also discussed. We mostly cover findings in rodents, but we also make special remarks regarding humans. We anticipate that this information will provide a framework for future research in the field.
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Affiliation(s)
- Mingxia Yao
- Center for Reproductive Health, School of Pharmaceutical Sciences, Hangzhou Medical College (Zhejiang Academy of Medical Sciences), Hangzhou, China
| | - Haoyang Qu
- Center for Reproductive Health, School of Pharmaceutical Sciences, Hangzhou Medical College (Zhejiang Academy of Medical Sciences), Hangzhou, China
| | - Yating Han
- Center for Reproductive Health, School of Pharmaceutical Sciences, Hangzhou Medical College (Zhejiang Academy of Medical Sciences), Hangzhou, China
| | - C Yan Cheng
- Department of Urology and Andrology, Sir Run-Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Xiang Xiao
- Center for Reproductive Health, School of Pharmaceutical Sciences, Hangzhou Medical College (Zhejiang Academy of Medical Sciences), Hangzhou, China.,Zhejiang Provincial Laboratory of Experimental Animal's & Nonclinical Laboratory Studies, Hangzhou Medical College, Hangzhou, China
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4
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Shan B, Liu Y, Yang C, Zhao Y, Sun D. Comparative transcriptomic analysis for identification of candidate sex-related genes and pathways in Crimson seabream (Parargyrops edita). Sci Rep 2021; 11:1077. [PMID: 33441831 PMCID: PMC7806868 DOI: 10.1038/s41598-020-80282-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Accepted: 12/18/2020] [Indexed: 01/29/2023] Open
Abstract
Teleost fishes display the largest array of sex-determining systems among animals, resulting in various reproductive strategies. Research on sex-related genes in teleosts will broaden our understanding of the process, and provide important insight into the plasticity of the sex determination process in vertebrates in general. Crimson seabream (Parargyrops edita Tanaka, 1916) is one of the most valuable and abundant fish resources throughout Asia. However, little genomic information on P. edita is available. In the present study, the transcriptomes of male and female P. edita were sequenced with RNA-seq technology. A total of 388,683,472 reads were generated from the libraries. After filtering and assembling, a total of 79,775 non redundant unigenes were obtained with an N50 of 2,921 bp. The unigenes were annotated with multiple public databases, including NT (53,556, 67.13%), NR (54,092, 67.81%), Swiss-Prot (45,265, 56.74%), KOG (41,274, 51.74%), KEGG (46,302, 58.04%), and GO (11,056, 13.86%) databases. Comparison of the unigenes of different sexes of P. edita revealed that 11,676 unigenes (9,335 in females, 2,341 in males) were differentially expressed between males and females. Of these, 5,463 were specifically expressed in females, and 1,134 were specifically expressed in males. In addition, the expression levels of ten unigenes were confirmed to validate the transcriptomic data by qRT-PCR. Moreover, 34,473 simple sequence repeats (SSRs) were identified in SSR-containing sequences, and 50 loci were randomly selected for primer development. Of these, 36 loci were successfully amplified, and 19 loci were polymorphic. Finally, our comparative analysis identified many sex-related genes (zps, amh, gsdf, sox4, cyp19a, etc.) and pathways (MAPK signaling pathway, p53 signaling pathway, etc.) of P. edita. This informative transcriptomic analysis provides valuable data to increase genomic resources of P. edita. The results will be useful for clarifying the molecular mechanism of sex determination and for future functional analyses of sex-associated genes.
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Affiliation(s)
- Binbin Shan
- Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture Rural Affairs, Guangzhou, China
- Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, China
- South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, China
| | - Yan Liu
- Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture Rural Affairs, Guangzhou, China
- Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, China
- South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, China
| | - Changping Yang
- Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture Rural Affairs, Guangzhou, China
- Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, China
- South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, China
| | - Yu Zhao
- Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture Rural Affairs, Guangzhou, China
- Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, China
- South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, China
| | - Dianrong Sun
- Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture Rural Affairs, Guangzhou, China.
- Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou, China.
- South China Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, China.
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5
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Reilly ML, Benmerah A. Ciliary kinesins beyond IFT: Cilium length, disassembly, cargo transport and signalling. Biol Cell 2019; 111:79-94. [PMID: 30720881 DOI: 10.1111/boc.201800074] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2018] [Accepted: 01/18/2019] [Indexed: 02/06/2023]
Abstract
Cilia and flagella are microtubule-based antenna which are highly conserved among eukaryotes. In vertebrates, primary and motile cilia have evolved to exert several key functions during development and tissue homoeostasis. Ciliary dysfunction in humans causes a highly heterogeneous group of diseases called ciliopathies, a class of genetic multisystemic disorders primarily affecting kidney, skeleton, retina, lung and the central nervous system. Among key ciliary proteins, kinesin family members (KIF) are microtubule-interacting proteins involved in many diverse cellular functions, including transport of cargo (organelles, proteins and lipids) along microtubules and regulating the dynamics of cytoplasmic and spindle microtubules through their depolymerising activity. Many KIFs are also involved in diverse ciliary functions including assembly/disassembly, motility and signalling. We here review these ciliary kinesins in vertebrates and focus on their involvement in ciliopathy-related disorders.
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Affiliation(s)
- Madeline Louise Reilly
- Laboratory of Hereditary Kidney Diseases, INSERM UMR 1163, Paris Descartes University, Imagine Institute, Paris, 75015, France.,Paris Diderot University, Paris, 75013, France
| | - Alexandre Benmerah
- Laboratory of Hereditary Kidney Diseases, INSERM UMR 1163, Paris Descartes University, Imagine Institute, Paris, 75015, France
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6
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Mason-Osann E, Dai A, Floro J, Lock YJ, Reiss M, Gali H, Matschulat A, Labadorf A, Flynn RL. Identification of a novel gene fusion in ALT positive osteosarcoma. Oncotarget 2018; 9:32868-32880. [PMID: 30214690 PMCID: PMC6132345 DOI: 10.18632/oncotarget.26029] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Accepted: 08/15/2018] [Indexed: 01/13/2023] Open
Abstract
The Alternative Lengthening of Telomeres (ALT) pathway stimulates telomere elongation and prevents cellular senescence in approximately 60% of osteosarcoma. While the precise mechanism underlying activation of the ALT pathway is unclear, mutations in the chromatin remodeling protein ATRX, histone chaperone DAXX, and the histone variant H3.3 correlate with ALT status. ATRX and DAXX facilitate deposition of the histone variant H3.3 within heterochromatic regions suggesting that loss of ATRX, DAXX, and/or H3.3 lead to defects in the stability of telomeric heterochromatin. Genetic mutations in ATRX, DAXX, and H3.3 have been detected in ALT positive cancers, however, a subset of ALT samples show loss of ATRX or DAXX protein expression or localization without evidence of genetic alterations suggesting additional uncharacterized defects in ATRX/DAXX/H3.3 function. Here, using Next Generation Sequencing we identified a novel gene fusion event between DAXX and the kinesin motor protein, KIFC3, leading to the translation of a chimeric DAXX-KIFC3 fusion protein. Moreover, we demonstrate that the fusion of KIFC3 to DAXX causes defects in DAXX function likely promoting ALT activity. These data highlight a potentially unrecognized mechanism of DAXX inactivation in ALT positive osteosarcoma and provide rationale for thorough and comprehensive analyses of ATRX/DAXX/H3.3 proteins in ALT positive cancers.
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Affiliation(s)
- Emily Mason-Osann
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
| | - Anqi Dai
- BU Bioinformatics Hub, Boston University, Boston, MA 02118, USA
| | - Jess Floro
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
| | - Ying Jie Lock
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
| | - Matthew Reiss
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
| | - Himabindu Gali
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
| | - Adeline Matschulat
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Adam Labadorf
- BU Bioinformatics Hub, Boston University, Boston, MA 02118, USA
| | - Rachel Litman Flynn
- Departments of Pharmacology and Experimental Therapeutics, and Medicine Cancer Center, Boston University School of Medicine, Boston, MA 02118, USA
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7
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Di Gioia SA, Farinelli P, Letteboer SJF, Arsenijevic Y, Sharon D, Roepman R, Rivolta C. Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network. Hum Mol Genet 2015; 24:3359-71. [PMID: 25749990 DOI: 10.1093/hmg/ddv085] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2014] [Accepted: 03/04/2015] [Indexed: 11/13/2022] Open
Abstract
Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal disease.
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Affiliation(s)
| | - Pietro Farinelli
- Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland
| | - Stef J F Letteboer
- Department of Human Genetics and Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and
| | - Yvan Arsenijevic
- Unit of Gene Therapy and Stem Cell Biology, Jules-Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland
| | - Dror Sharon
- Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
| | - Ronald Roepman
- Department of Human Genetics and Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and
| | - Carlo Rivolta
- Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland
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8
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Hu JR, Liu M, Hou CC, She ZY, Wang DH, Hao SL, Zhang YP, Yang WX. Gene expression pattern of KIFC3 during spermatogenesis of the skink Eumeces chinensis. Gene 2014; 556:206-12. [PMID: 25434495 DOI: 10.1016/j.gene.2014.11.058] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 11/23/2014] [Accepted: 11/25/2014] [Indexed: 12/28/2022]
Abstract
Kinesin superfamily is a class of microtubule-dependent motors that play crucial roles in acrosome biogenesis, nuclear reshaping and flagellum formation during spermiogenesis. We have cloned kinesin-like gene kifc3 (termed ec-kifc3) from the total RNA of the testis of the skink Eumeces chinensis. The cDNA sequence of ec-kifc3 had a full-length of 3033bp, including a 260bp 5'-untranslated region (5'UTR), a 445bp 3'-untranslated region (3'UTR) and an open reading frame that encoded a 775-amino-acid protein. Additionally, the calculated molecular weight of the putative ec-KIFC3 was 87kDa and its estimated isoelectric point was 6.18. Structurally, the putative ec-KIFC3 had three domains: head domain, neck domain and tail domain. Protein alignment demonstrated that ec-KIFC3 had 47.2%, 67.8%, 68.8%, 69.3% and 76.8% identity with its homologues in Xenopus laevis, Mus musculus, Cricetulus griseus, Homo sapiens, and Gallus gallus. The phylogenetic analysis showed that ec-KIFC3 was more related to KIFC3 in vertebrates than invertebrates. Tissue expression results showed the presence of ec-KIFC3 in various tissues with its highest expression in the testis. In situ hybridization demonstrated that ec-KIFC3 mRNA was distributed around the nucleus in early and middle stage spermatids and expressed in the nucleus in the elongating spermatids during spermiogenesis. Besides, the ec-KIFC3 mRNA was expressed in the acrosome of the developmental spermatids. From the results of in situ hybridization and previous researches, we speculated that ec-KIFC3 may play a role in nuclear morphogenesis and acrosome formation during spermiogenesis of E. chinensis.
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Affiliation(s)
- Jian-Rao Hu
- College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, PR China
| | - Mei Liu
- College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, PR China; The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
| | - Cong-Cong Hou
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
| | - Zhen-Yu She
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
| | - Da-Hui Wang
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China
| | - Shuang-Li Hao
- College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, PR China
| | - Yong-Pu Zhang
- College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, PR China
| | - Wan-Xi Yang
- The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou 310058, PR China.
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9
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Nachbar J, Lázaro-Diéguez F, Prekeris R, Cohen D, Müsch A. KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis. Cell Cycle 2013; 13:426-33. [PMID: 24275865 DOI: 10.4161/cc.27266] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Kinesin-14 motor proteins play a variety of roles during metaphase and anaphase. However, it is not known whether members of this family of motors also participate in the dramatic changes in mitotic spindle organization during the transition from telophase to cytokinesis. We have identified the minus-end-directed motor, KIFC3, as an important contributor to central bridge morphology at this stage. KIFC3's unique motor-dependent localization at the central bridge allows it to congress microtubules, promoting efficient progress through cytokinesis. Conversely, when KIFC3 function is perturbed, abscission is delayed, and the central bridge is both widened and extended. Examination of KIFC3 on growing microtubules in interphase indicates that it caps microtubules released from the centrosome, both in the region of the centrosome and in the cell periphery. In line with other kinesin-14 family members, KIFC3 may guide free microtubules to their destination at the bridge and/or may slide and crosslink central bridge microtubules in order to stage the cells for abscission.
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Affiliation(s)
- Jeannette Nachbar
- Department of Developmental and Molecular Biology; Albert Einstein College of Medicine; New York, NY USA
| | - Francisco Lázaro-Diéguez
- Department of Developmental and Molecular Biology; Albert Einstein College of Medicine; New York, NY USA
| | | | - David Cohen
- Department of Developmental and Molecular Biology; Albert Einstein College of Medicine; New York, NY USA
| | - Anne Müsch
- Department of Developmental and Molecular Biology; Albert Einstein College of Medicine; New York, NY USA
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10
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Dietrich D, Seiler F, Essmann F, Dodt G. Identification of the kinesin KifC3 as a new player for positioning of peroxisomes and other organelles in mammalian cells. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2013; 1833:3013-3024. [PMID: 23954441 DOI: 10.1016/j.bbamcr.2013.08.002] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Revised: 07/19/2013] [Accepted: 08/02/2013] [Indexed: 01/25/2023]
Abstract
The attachment of organelles to the cytoskeleton and directed organelle transport is essential for cellular morphology and function. In contrast to other cell organelles like the endoplasmic reticulum or the Golgi apparatus, peroxisomes are evenly distributed in the cytoplasm, which is achieved by binding of peroxisomes to microtubules and their bidirectional transport by the microtubule motor proteins kinesin-1 (Kif5) and cytoplasmic dynein. KifC3, belonging to the group of C-terminal kinesins, has been identified to interact with the human peroxin PEX1 in a yeast two-hybrid screen. We investigated the potential involvement of KifC3 in peroxisomal transport. Interaction of KifC3 and the AAA-protein (ATPase associated with various cellular activities) PEX1 was confirmed by in vivo colocalization and by coimmunoprecipitation from cell lysates. Furthermore, knockdown of KifC3 using RNAi resulted in an increase of cells with perinuclear-clustered peroxisomes, indicating enhanced minus-end directed motility of peroxisomes. The occurrence of this peroxisomal phenotype was cell cycle phase independent, while microtubules were essential for phenotype formation. We conclude that KifC3 may play a regulatory role in minus-end directed peroxisomal transport for example by blocking the motor function of dynein at peroxisomes. Knockdown of KifC3 would then lead to increased minus-end directed peroxisomal transport and cause the observed peroxisomal clustering at the microtubule-organizing center.
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Affiliation(s)
- Denise Dietrich
- Interfaculty Institute of Biochemistry, Cell Biochemistry, University of Tuebingen, D-72076 Tuebingen, Germany
| | - Florian Seiler
- Interfaculty Institute of Biochemistry, Cell Biochemistry, University of Tuebingen, D-72076 Tuebingen, Germany
| | - Frank Essmann
- Interfaculty Institute of Biochemistry, Molecular Medicine, University of Tuebingen, D-72076 Tuebingen, Germany
| | - Gabriele Dodt
- Interfaculty Institute of Biochemistry, Cell Biochemistry, University of Tuebingen, D-72076 Tuebingen, Germany.
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11
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Zaichick SV, Bohannon KP, Smith GA. Alphaherpesviruses and the cytoskeleton in neuronal infections. Viruses 2011; 3:941-81. [PMID: 21994765 PMCID: PMC3185784 DOI: 10.3390/v3070941] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2011] [Revised: 06/03/2011] [Accepted: 06/17/2011] [Indexed: 12/13/2022] Open
Abstract
Following infection of exposed peripheral tissues, neurotropic alphaherpesviruses invade nerve endings and deposit their DNA genomes into the nuclei of neurons resident in ganglia of the peripheral nervous system. The end result of these events is the establishment of a life-long latent infection. Neuroinvasion typically requires efficient viral transmission through a polarized epithelium followed by long-distance transport through the viscous axoplasm. These events are mediated by the recruitment of the cellular microtubule motor proteins to the intracellular viral particle and by alterations to the cytoskeletal architecture. The focus of this review is the interplay between neurotropic herpesviruses and the cytoskeleton.
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Affiliation(s)
- Sofia V Zaichick
- Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
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12
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Hirokawa N, Niwa S, Tanaka Y. Molecular Motors in Neurons: Transport Mechanisms and Roles in Brain Function, Development, and Disease. Neuron 2010; 68:610-38. [DOI: 10.1016/j.neuron.2010.09.039] [Citation(s) in RCA: 831] [Impact Index Per Article: 55.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/22/2010] [Indexed: 12/11/2022]
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13
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Thorsteinsson RI, Christensen ST, Pedersen LB. Using quantitative PCR to identify kinesin-3 genes that are upregulated during growth arrest in mouse NIH3T3 cells. Methods Cell Biol 2010; 94:67-86. [PMID: 20362085 DOI: 10.1016/s0091-679x(08)94003-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
Most cells in our body form a single primary cilium when entering growth arrest. During the past decade, a number of studies have revealed a key role for primary cilia in coordinating a variety of signaling pathways that control important cellular and developmental processes. Consequently, significant effort has been directed toward the identification of genes involved in ciliary assembly and function. Many candidate ciliary genes and proteins have been identified using large-scale "omics" approaches, including proteomics, transcriptomics, and comparative genomics. Although such large-scale approaches can be extremely informative, additional validation of candidate ciliary genes using alternative "small-scale" approaches is often necessary. Here we describe a quantitative PCR-based method that can be used to screen groups of genes for those that are upregulated during growth arrest in cultured mouse NIH3T3 cells and those that might have cilia-related functions. We employed this method to specifically search for mouse kinesin-3 genes that are upregulated during growth arrest and identified three such genes (Kif13A, Kif13B, and Kif16A). In principle, however, the method can be extended to identify other genes or gene families that are upregulated during growth arrest.
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Affiliation(s)
- Rikke I Thorsteinsson
- Department of Biology, Section of Cell and Developmental Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen OE, Denmark
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De S, Cipriano R, Jackson MW, Stark GR. Overexpression of kinesins mediates docetaxel resistance in breast cancer cells. Cancer Res 2009; 69:8035-42. [PMID: 19789344 DOI: 10.1158/0008-5472.can-09-1224] [Citation(s) in RCA: 109] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumor-derived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes.
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Affiliation(s)
- Sarmishtha De
- Department of Genetics and Pathology, Case Western Reserve University, Case Comprehensive Cancer Center, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
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Nath S, Bananis E, Sarkar S, Stockert RJ, Sperry AO, Murray JW, Wolkoff AW. Kif5B and Kifc1 interact and are required for motility and fission of early endocytic vesicles in mouse liver. Mol Biol Cell 2007; 18:1839-49. [PMID: 17360972 PMCID: PMC1855015 DOI: 10.1091/mbc.e06-06-0524] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5'-adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.
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Affiliation(s)
- Sangeeta Nath
- Department of Anatomy and Structural Biology and
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
| | - Eustratios Bananis
- Department of Anatomy and Structural Biology and
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
| | - Souvik Sarkar
- Department of Anatomy and Structural Biology and
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
| | - Richard J. Stockert
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
| | - Ann O. Sperry
- Department of Anatomy and Cell Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27858
| | - John W. Murray
- Department of Anatomy and Structural Biology and
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
| | - Allan W. Wolkoff
- Department of Anatomy and Structural Biology and
- *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461; and
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Chevalier-Larsen E, Holzbaur ELF. Axonal transport and neurodegenerative disease. Biochim Biophys Acta Mol Basis Dis 2006; 1762:1094-108. [PMID: 16730956 DOI: 10.1016/j.bbadis.2006.04.002] [Citation(s) in RCA: 328] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2006] [Revised: 03/24/2006] [Accepted: 04/11/2006] [Indexed: 01/12/2023]
Abstract
Neurons have extensive processes and communication between those processes and the cell body is crucial to neuronal function and survival. Thus, neurons are uniquely dependent on microtubule based transport. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases. We describe the motor, cytoskeletal, and adaptor proteins involved in axonal transport and their interactions. Data linking disruption of axonal transport to diseases such as ALS are discussed. Finally, we explore the pathways that may cause neuronal dysfunction and death.
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17
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Abstract
Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.
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Affiliation(s)
- Matthew A Berezuk
- Department of Biology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA
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18
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Abstract
After internalization, endocytic material is actively transported through the cytoplasm, predominantly by microtubule motor proteins. Microtubule-based endocytic transport facilitates sorting of endocytic contents, vesicle fusion and fission, delivery to lysosomes, cytosolic dispersal, as well as nuclear uptake and cytosolic egress of pathogens. Endosomes, like most organelles, move bidirectionally through the cytosol and regulate their cellular location by controlling the activity of motor proteins, and potentially by controlling microtubule and actin polymerization. Control of motor protein activity is manifest by increased microtubule "run lengths", and the binding of motor proteins to organelles can be regulated by motor protein receptors. A mechanistic understanding of how organelles control motor protein activity to allow for endocytic sorting presents an exciting avenue for future research.
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Affiliation(s)
- John W Murray
- Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 517 Ullmann Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
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Xu Y, Takeda S, Nakata T, Noda Y, Tanaka Y, Hirokawa N. Role of KIFC3 motor protein in Golgi positioning and integration. J Cell Biol 2002; 158:293-303. [PMID: 12135985 PMCID: PMC2173137 DOI: 10.1083/jcb.200202058] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.
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Affiliation(s)
- Ying Xu
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan
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DeGiorgis JA, Reese TS, Bearer EL. Association of a nonmuscle myosin II with axoplasmic organelles. Mol Biol Cell 2002; 13:1046-57. [PMID: 11907281 PMCID: PMC99618 DOI: 10.1091/mbc.01-06-0315] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a approximately 220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this approximately 220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single approximately 220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor.
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Affiliation(s)
- Joseph A DeGiorgis
- Molecular & Cell Biology and Biochemistry Program, Brown University, Providence, RI 02912, USA
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Noda Y, Okada Y, Saito N, Setou M, Xu Y, Zhang Z, Hirokawa N. KIFC3, a microtubule minus end-directed motor for the apical transport of annexin XIIIb-associated Triton-insoluble membranes. J Cell Biol 2001; 155:77-88. [PMID: 11581287 PMCID: PMC2150803 DOI: 10.1083/jcb.200108042] [Citation(s) in RCA: 129] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We have identified and characterized a COOH-terminal motor domain-type kinesin superfamily protein (KIFC), KIFC3, in the kidney. KIFC3 is a minus end-directed microtubule motor protein, therefore it accumulates in regions where minus ends of microtubules assemble. In polarized epithelial cells, KIFC3 is localized on membrane organelles immediately beneath the apical plasma membrane of renal tubular epithelial cells in vivo and polarized MDCK II cells in vitro. Flotation assay, coupled with detergent extraction, demonstrated that KIFC3 is associated with Triton X-100-insoluble membrane organelles, and that it overlaps with apically transported TGN-derived vesicles. This was confirmed by immunoprecipitation and by GST pulldown experiments showing the specific colocalization of KIFC3 and annexin XIIIb, a previously characterized membrane protein for apically transported vesicles (Lafont, F., S. Lecat, P. Verkade, and K. Simons. 1998. J. Cell Biol. 142:1413-1427). Furthermore, we proved that the apical transport of both influenza hemagglutinin and annexin XIIIb was partially inhibited or accelerated by overexpression of motor-domainless (dominant negative) or full-length KIFC3, respectively. Absence of cytoplasmic dynein on these annexin XIIIb-associated vesicles and distinct distribution of the two motors on the EM level verified the existence of KIFC3-driven transport in epithelial cells.
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Affiliation(s)
- Y Noda
- Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Tokyo 113-0033, Japan
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Abstract
Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. In the mouse, several kinesin motors have been characterized and are suggested to play roles in axonal and/or dendritic transport. One such kinesin is KifC2. Sequence and secondary structure analysis revealed that KifC2 is a member of the C-terminal motor family. Northern and Western blot analyses indicated that KifC2 is specifically expressed in both the central and peripheral nervous systems. The cellular locations of the KifC2 proteins were found to be mainly in neural cell bodies and dendrites but also in axons. To understand the in vivo function of the KifC2 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC2 gene. Homozygous KifC2 mutants were viable and reproduced normally, and their development was apparently normal. These results suggest that KifC2 is dispensable for normal neural development and behavior in the mouse.
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Affiliation(s)
- Z Yang
- Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California San Diego, La Jolla, California 92093, USA
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