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An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis.

Kaur S, Fielding AB, Gassner G, Carter NJ, Royle SJ - Elife (2014)

Bottom Line: In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME.However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment.Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be 'restarted' in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown. DOI: http://dx.doi.org/10.7554/eLife.00829.001.

No MeSH data available.


Related in: MedlinePlus

Mitotic phosphorylation does not explain mitotic shutdown of CME.(A) Western blot to show Dab2 phosphorylation and inhibition by the Cdk1 inhibitor flavopiridol (5 µM, 10 min). Interphase or mitotic cell lysates prepared from cells expressing GFP or GFP-Rap(Q63E) to restart CME in mitotic cells. Blots were probed for Dab2 and β-tubulin as a loading control, or Cyclin-B1 and Cdk1. (B) Bar chart to show the average transferrin uptake in interphase (orange) or mitotic (purple) populations of cells by flow cytometry. The mean ± SEM of three separate experiments are shown.DOI:http://dx.doi.org/10.7554/eLife.00829.010
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fig6: Mitotic phosphorylation does not explain mitotic shutdown of CME.(A) Western blot to show Dab2 phosphorylation and inhibition by the Cdk1 inhibitor flavopiridol (5 µM, 10 min). Interphase or mitotic cell lysates prepared from cells expressing GFP or GFP-Rap(Q63E) to restart CME in mitotic cells. Blots were probed for Dab2 and β-tubulin as a loading control, or Cyclin-B1 and Cdk1. (B) Bar chart to show the average transferrin uptake in interphase (orange) or mitotic (purple) populations of cells by flow cytometry. The mean ± SEM of three separate experiments are shown.DOI:http://dx.doi.org/10.7554/eLife.00829.010

Mentions: An alternative explanation for CME shutdown in mitosis is the phosphorylation of endocytic proteins by mitotic kinases (Pypaert et al., 1991; Chen et al., 1999; Fielding and Royle, 2013). Our observations, that mitotic cells are competent for CME if the actin cytoskeleton is made available, runs counter to this hypothesis. We first tested if mitotic phosphorylation of endocytic proteins was maintained in cells with restored CME. We analysed the phosphorylation status of Dab2 by western blotting. Dab2 is a clathrin adaptor for cargo proteins with an NPXY endocytic motif (Mishra et al., 2002) and it is known to be phosphorylated in mitosis (He et al., 2003; Chetrit et al., 2011). Figure 6A shows that Dab2 is phosphorylated in mitosis and that this phosphorylation can be reversed by brief treatment of mitotic cells with the Cdk1 inhibitor flavopiridol (5 µM, 10 min) (Losiewicz et al., 1994). An identical situation was seen in lysates from cells expressing GFP-Rap1(Q63E) (Figure 6A). This suggests that CME of proteins with NPXY motifs can occur (Figure 5) despite maintained mitotic phosphorylation of Dab2 by Cdk1. Secondly, we tested whether inhibition of Cdk1 was sufficient to restart CME. Such restarting would be expected if mitotic phosphorylation inhibited the CME machinery directly. To do this, CME was measured using transferrin uptake and flow cytometry. The brief incubation of mitotic cells with flavopiridol (5 or 10 µM) did not restart CME. Together, these results indicate that the direct mitotic phosphorylation of endocytic proteins cannot account for mitotic shut down of CME. It remains possible that phosphorylation of endocytic proteins may have a more minor, modulatory role in mitotic shutdown of CME, but we found no evidence that phosphorylation of the CME machinery renders it inactive. Moreover, it should be noted that cyclin B1-Cdk1 activity orchestrates mitosis and therefore drives the changes that result in the inhibition of CME at a higher level.10.7554/eLife.00829.010Figure 6.Mitotic phosphorylation does not explain mitotic shutdown of CME.


An unmet actin requirement explains the mitotic inhibition of clathrin-mediated endocytosis.

Kaur S, Fielding AB, Gassner G, Carter NJ, Royle SJ - Elife (2014)

Mitotic phosphorylation does not explain mitotic shutdown of CME.(A) Western blot to show Dab2 phosphorylation and inhibition by the Cdk1 inhibitor flavopiridol (5 µM, 10 min). Interphase or mitotic cell lysates prepared from cells expressing GFP or GFP-Rap(Q63E) to restart CME in mitotic cells. Blots were probed for Dab2 and β-tubulin as a loading control, or Cyclin-B1 and Cdk1. (B) Bar chart to show the average transferrin uptake in interphase (orange) or mitotic (purple) populations of cells by flow cytometry. The mean ± SEM of three separate experiments are shown.DOI:http://dx.doi.org/10.7554/eLife.00829.010
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3924242&req=5

fig6: Mitotic phosphorylation does not explain mitotic shutdown of CME.(A) Western blot to show Dab2 phosphorylation and inhibition by the Cdk1 inhibitor flavopiridol (5 µM, 10 min). Interphase or mitotic cell lysates prepared from cells expressing GFP or GFP-Rap(Q63E) to restart CME in mitotic cells. Blots were probed for Dab2 and β-tubulin as a loading control, or Cyclin-B1 and Cdk1. (B) Bar chart to show the average transferrin uptake in interphase (orange) or mitotic (purple) populations of cells by flow cytometry. The mean ± SEM of three separate experiments are shown.DOI:http://dx.doi.org/10.7554/eLife.00829.010
Mentions: An alternative explanation for CME shutdown in mitosis is the phosphorylation of endocytic proteins by mitotic kinases (Pypaert et al., 1991; Chen et al., 1999; Fielding and Royle, 2013). Our observations, that mitotic cells are competent for CME if the actin cytoskeleton is made available, runs counter to this hypothesis. We first tested if mitotic phosphorylation of endocytic proteins was maintained in cells with restored CME. We analysed the phosphorylation status of Dab2 by western blotting. Dab2 is a clathrin adaptor for cargo proteins with an NPXY endocytic motif (Mishra et al., 2002) and it is known to be phosphorylated in mitosis (He et al., 2003; Chetrit et al., 2011). Figure 6A shows that Dab2 is phosphorylated in mitosis and that this phosphorylation can be reversed by brief treatment of mitotic cells with the Cdk1 inhibitor flavopiridol (5 µM, 10 min) (Losiewicz et al., 1994). An identical situation was seen in lysates from cells expressing GFP-Rap1(Q63E) (Figure 6A). This suggests that CME of proteins with NPXY motifs can occur (Figure 5) despite maintained mitotic phosphorylation of Dab2 by Cdk1. Secondly, we tested whether inhibition of Cdk1 was sufficient to restart CME. Such restarting would be expected if mitotic phosphorylation inhibited the CME machinery directly. To do this, CME was measured using transferrin uptake and flow cytometry. The brief incubation of mitotic cells with flavopiridol (5 or 10 µM) did not restart CME. Together, these results indicate that the direct mitotic phosphorylation of endocytic proteins cannot account for mitotic shut down of CME. It remains possible that phosphorylation of endocytic proteins may have a more minor, modulatory role in mitotic shutdown of CME, but we found no evidence that phosphorylation of the CME machinery renders it inactive. Moreover, it should be noted that cyclin B1-Cdk1 activity orchestrates mitosis and therefore drives the changes that result in the inhibition of CME at a higher level.10.7554/eLife.00829.010Figure 6.Mitotic phosphorylation does not explain mitotic shutdown of CME.

Bottom Line: In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME.However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment.Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is the major internalisation route for many different receptor types in mammalian cells. CME is shut down during early mitosis, but the mechanism of this inhibition is unclear. In this study, we show that the mitotic shutdown is due to an unmet requirement for actin in CME. In mitotic cells, membrane tension is increased and this invokes a requirement for the actin cytoskeleton to assist the CME machinery to overcome the increased load. However, the actin cytoskeleton is engaged in the formation of a rigid cortex in mitotic cells and is therefore unavailable for deployment. We demonstrate that CME can be 'restarted' in mitotic cells despite high membrane tension, by allowing actin to engage in endocytosis. Mitotic phosphorylation of endocytic proteins is maintained in mitotic cells with restored CME, indicating that direct phosphorylation of the CME machinery does not account for shutdown. DOI: http://dx.doi.org/10.7554/eLife.00829.001.

No MeSH data available.


Related in: MedlinePlus