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Clathrin Is Spindle-Associated but Not Essential for Mitosis

BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical...

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Autores principales: Borlido, Joana, Veltri, Greg, Jackson, Antony P., Mills, Ian G.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2518958/
https://www.ncbi.nlm.nih.gov/pubmed/18769625
http://dx.doi.org/10.1371/journal.pone.0003115
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author Borlido, Joana
Veltri, Greg
Jackson, Antony P.
Mills, Ian G.
author_facet Borlido, Joana
Veltri, Greg
Jackson, Antony P.
Mills, Ian G.
author_sort Borlido, Joana
collection PubMed
description BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis. METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression. CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells.
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spelling pubmed-25189582008-09-03 Clathrin Is Spindle-Associated but Not Essential for Mitosis Borlido, Joana Veltri, Greg Jackson, Antony P. Mills, Ian G. PLoS One Research Article BACKGROUND: Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis. METHODOLOGY/PRINCIPAL FINDINGS: Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression. CONCLUSIONS/SIGNIFICANCE: We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells. Public Library of Science 2008-09-03 /pmc/articles/PMC2518958/ /pubmed/18769625 http://dx.doi.org/10.1371/journal.pone.0003115 Text en Borlido et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Borlido, Joana
Veltri, Greg
Jackson, Antony P.
Mills, Ian G.
Clathrin Is Spindle-Associated but Not Essential for Mitosis
title Clathrin Is Spindle-Associated but Not Essential for Mitosis
title_full Clathrin Is Spindle-Associated but Not Essential for Mitosis
title_fullStr Clathrin Is Spindle-Associated but Not Essential for Mitosis
title_full_unstemmed Clathrin Is Spindle-Associated but Not Essential for Mitosis
title_short Clathrin Is Spindle-Associated but Not Essential for Mitosis
title_sort clathrin is spindle-associated but not essential for mitosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2518958/
https://www.ncbi.nlm.nih.gov/pubmed/18769625
http://dx.doi.org/10.1371/journal.pone.0003115
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