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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

Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.(A) Confocal micrographs of a HeLa cell expressing GFP-tagged clathrin light chain a (clathrin, green). The same cell is shown before and after application of transferrin-Alexa568 (Tf, red). A single XY section is shown together with a YZ view through the cell centre (right). Far right, 3D projection of the whole confocal series. (B) Schematic diagram of the purification and proteomic analysis of clathrin-coated structures purified from cells in interphase or metaphase. (C) Bar chart to show the comparative interphase/mitotic abundance for CCV proteins from the search list. The interphase or mitotic LFQ value for each protein derived from four separate experiments were compared. Red bars show proteins related to the actin cytoskeleton. Red circles indicate proteins verified in D. Inset: histogram to show the frequency of abundances for all proteins in the analysis. A single Gaussian function was fitted to the data, the mean and variance of which is shown in the main bar chart. Dotted line shows the average comparative abundance and the shaded area represents ± 2 standard deviations. (D) Representative fluorescence micrographs to show the colocalisation of GFP-tagged clathrin light chain a (clathrin, green) in interphase or mitosis, with HIP1R-tDimer-RFP, tdTomato-HIP1 or mCherry-cortactin (red). Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.00829.004
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fig2: Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.(A) Confocal micrographs of a HeLa cell expressing GFP-tagged clathrin light chain a (clathrin, green). The same cell is shown before and after application of transferrin-Alexa568 (Tf, red). A single XY section is shown together with a YZ view through the cell centre (right). Far right, 3D projection of the whole confocal series. (B) Schematic diagram of the purification and proteomic analysis of clathrin-coated structures purified from cells in interphase or metaphase. (C) Bar chart to show the comparative interphase/mitotic abundance for CCV proteins from the search list. The interphase or mitotic LFQ value for each protein derived from four separate experiments were compared. Red bars show proteins related to the actin cytoskeleton. Red circles indicate proteins verified in D. Inset: histogram to show the frequency of abundances for all proteins in the analysis. A single Gaussian function was fitted to the data, the mean and variance of which is shown in the main bar chart. Dotted line shows the average comparative abundance and the shaded area represents ± 2 standard deviations. (D) Representative fluorescence micrographs to show the colocalisation of GFP-tagged clathrin light chain a (clathrin, green) in interphase or mitosis, with HIP1R-tDimer-RFP, tdTomato-HIP1 or mCherry-cortactin (red). Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.00829.004

Mentions: Clathrin-coated structures (CCSs, pits, and vesicles) are present in mitotic cells with the pits being arrested at the cell surface in a shallow state (Pypaert et al., 1987). Fluorescent transferrin binding to abundant transferrin receptors on the plasma membrane can be seen in these pits but the uptake of transferrin is prevented (Figure 2A; Fielding et al., 2012). Our hypothesis was that differences in the proteomes of CCSs purified from interphase or mitotic cells would explain why clathrin-coated pits are arrested at the surface. For example, components of the CME machinery that are regulated might be present in interphase CCSs but could be less abundant in mitotic CCSs. To identify such differences, we prepared fractions enriched in CCSs from interphase or mitotic HeLa cells, according to established methods (Borner et al., 2006, 2012). These fractions were analysed by mass spectrometry and label-free quantitation (Figure 2B). Over four independent experiments, we compared the relative abundance of 1253 proteins, only a subset of which are confirmed CCS proteins (Borner et al., 2006, 2012). The list of all abundances was used to characterise the variance of the dataset and identify outliers (‘Materials and methods’). We found that cortactin was the protein most consistently reduced in mitotic CCSs compared to interphase (LFQ intensity ratio = 46.2). Cortactin is an activator of Arp2/3-dependent actin polymerisation. A list of bona fide CCS proteins (Borner et al., 2012) was therefore supplemented with components of the actin cytoskeleton that are recruited to CCSs (Taylor et al., 2011). The relative abundance of these proteins is shown in Figure 2C. These data show that most of the core CME machinery is not altered significantly between CCS-containing fractions from interphase and mitotic samples. Consistent with previous results (Chetrit et al., 2011; Kozik et al., 2013), Dab2 was less abundant and PICALM more abundant in mitotic fractions. HIP1 and HIP1R appeared to be differentially regulated. These two proteins link the clathrin machinery with the actin cytoskeleton and are regulated by binding clathrin light chain (Le Clainche et al., 2007; Wilbur et al., 2008). The accumulation of HIP1R and the absence of cortactin in mitotic fractions were interesting, given that these two proteins have been shown previously to be coupled functionally (Le Clainche et al., 2007). Other interesting differences, to be explored in the future, included the accumulation of NSF (N-Ethylmaleimide-Sensitive Factor) in mitotic CCSs (LFQ intensity ratio = 0.077).10.7554/eLife.00829.004Figure 2.Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.


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

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

Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.(A) Confocal micrographs of a HeLa cell expressing GFP-tagged clathrin light chain a (clathrin, green). The same cell is shown before and after application of transferrin-Alexa568 (Tf, red). A single XY section is shown together with a YZ view through the cell centre (right). Far right, 3D projection of the whole confocal series. (B) Schematic diagram of the purification and proteomic analysis of clathrin-coated structures purified from cells in interphase or metaphase. (C) Bar chart to show the comparative interphase/mitotic abundance for CCV proteins from the search list. The interphase or mitotic LFQ value for each protein derived from four separate experiments were compared. Red bars show proteins related to the actin cytoskeleton. Red circles indicate proteins verified in D. Inset: histogram to show the frequency of abundances for all proteins in the analysis. A single Gaussian function was fitted to the data, the mean and variance of which is shown in the main bar chart. Dotted line shows the average comparative abundance and the shaded area represents ± 2 standard deviations. (D) Representative fluorescence micrographs to show the colocalisation of GFP-tagged clathrin light chain a (clathrin, green) in interphase or mitosis, with HIP1R-tDimer-RFP, tdTomato-HIP1 or mCherry-cortactin (red). Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.00829.004
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fig2: Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.(A) Confocal micrographs of a HeLa cell expressing GFP-tagged clathrin light chain a (clathrin, green). The same cell is shown before and after application of transferrin-Alexa568 (Tf, red). A single XY section is shown together with a YZ view through the cell centre (right). Far right, 3D projection of the whole confocal series. (B) Schematic diagram of the purification and proteomic analysis of clathrin-coated structures purified from cells in interphase or metaphase. (C) Bar chart to show the comparative interphase/mitotic abundance for CCV proteins from the search list. The interphase or mitotic LFQ value for each protein derived from four separate experiments were compared. Red bars show proteins related to the actin cytoskeleton. Red circles indicate proteins verified in D. Inset: histogram to show the frequency of abundances for all proteins in the analysis. A single Gaussian function was fitted to the data, the mean and variance of which is shown in the main bar chart. Dotted line shows the average comparative abundance and the shaded area represents ± 2 standard deviations. (D) Representative fluorescence micrographs to show the colocalisation of GFP-tagged clathrin light chain a (clathrin, green) in interphase or mitosis, with HIP1R-tDimer-RFP, tdTomato-HIP1 or mCherry-cortactin (red). Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.00829.004
Mentions: Clathrin-coated structures (CCSs, pits, and vesicles) are present in mitotic cells with the pits being arrested at the cell surface in a shallow state (Pypaert et al., 1987). Fluorescent transferrin binding to abundant transferrin receptors on the plasma membrane can be seen in these pits but the uptake of transferrin is prevented (Figure 2A; Fielding et al., 2012). Our hypothesis was that differences in the proteomes of CCSs purified from interphase or mitotic cells would explain why clathrin-coated pits are arrested at the surface. For example, components of the CME machinery that are regulated might be present in interphase CCSs but could be less abundant in mitotic CCSs. To identify such differences, we prepared fractions enriched in CCSs from interphase or mitotic HeLa cells, according to established methods (Borner et al., 2006, 2012). These fractions were analysed by mass spectrometry and label-free quantitation (Figure 2B). Over four independent experiments, we compared the relative abundance of 1253 proteins, only a subset of which are confirmed CCS proteins (Borner et al., 2006, 2012). The list of all abundances was used to characterise the variance of the dataset and identify outliers (‘Materials and methods’). We found that cortactin was the protein most consistently reduced in mitotic CCSs compared to interphase (LFQ intensity ratio = 46.2). Cortactin is an activator of Arp2/3-dependent actin polymerisation. A list of bona fide CCS proteins (Borner et al., 2012) was therefore supplemented with components of the actin cytoskeleton that are recruited to CCSs (Taylor et al., 2011). The relative abundance of these proteins is shown in Figure 2C. These data show that most of the core CME machinery is not altered significantly between CCS-containing fractions from interphase and mitotic samples. Consistent with previous results (Chetrit et al., 2011; Kozik et al., 2013), Dab2 was less abundant and PICALM more abundant in mitotic fractions. HIP1 and HIP1R appeared to be differentially regulated. These two proteins link the clathrin machinery with the actin cytoskeleton and are regulated by binding clathrin light chain (Le Clainche et al., 2007; Wilbur et al., 2008). The accumulation of HIP1R and the absence of cortactin in mitotic fractions were interesting, given that these two proteins have been shown previously to be coupled functionally (Le Clainche et al., 2007). Other interesting differences, to be explored in the future, included the accumulation of NSF (N-Ethylmaleimide-Sensitive Factor) in mitotic CCSs (LFQ intensity ratio = 0.077).10.7554/eLife.00829.004Figure 2.Comparative proteomics of fractions containing clathrin-coated membranes purified from interphase and mitotic HeLa cells.

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