Limits...
A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

Show MeSH
Synthetic septation phenotypes observed in double mutants in genes encoding ESCRT proteins and (a) plo1-ts35, (b) ark1-T8 or (c) clp1Δ.Fission yeast double mutant strains were grown in complete liquid medium at 25°C to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Scale bar, 10 µm. The frequency of phenotypes A–F (described in Figure 1a) was quantitatively analysed in strains containing double mutants, in comparison to each parent. In each case 400 cells were counted in triplicate (*p<0.05). Each of the ESCRT genes labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214795&req=5

pone-0111789-g002: Synthetic septation phenotypes observed in double mutants in genes encoding ESCRT proteins and (a) plo1-ts35, (b) ark1-T8 or (c) clp1Δ.Fission yeast double mutant strains were grown in complete liquid medium at 25°C to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Scale bar, 10 µm. The frequency of phenotypes A–F (described in Figure 1a) was quantitatively analysed in strains containing double mutants, in comparison to each parent. In each case 400 cells were counted in triplicate (*p<0.05). Each of the ESCRT genes labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).

Mentions: To further understand the relationship between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase, double mutants were examined for potential septal defects. Double mutants were compared to both sets of respective mutant parents, and the septation profiles for plo1-ts35, clp1Δ, ark1-T8 and ark1-T11 single mutants relative to wild-type have been shown in Fig. S3. A higher proportion of phenotype Classes C–F was observed in plo1-ts35 cells, and of Classes C and F in clp1Δ cells (Fig. S3). Interestingly, plo1 mutants when combined with individual mutations in ESCRT genes frequently revealed a decreased proportion of the Class F phenotype, indicative of phenotypic rescue and the interaction of these genes within a shared pathway. For instance, the vps4Δ strain exhibited the Class F phenotype in 39% of cells; this was reduced to 23% in plo1-ts35 vps4Δ cells (p<0.05 relative to both parents; Fig. 2a). Both sets of double mutants also exhibited increases in phenotype Classes B–E (Fig. 2a and 2c). An increased proportion of Class B, E and F was observed in ark1-T8 cells (Fig. S3), and analysis of double mutants revealed a higher prevalence of Classes B in particular (Fig. 2b). A similar increase in Class B was observed in double mutants with ark1-T11 (Fig. S4). Collectively, these data are highly suggestive of genetic interactions between ESCRT and plo1, ark1 and cdc14 genes, and imply cooperation of these genes to control cell separation in this organism.


A complex network of interactions between mitotic kinases, phosphatases and ESCRT proteins regulates septation and membrane trafficking in S. pombe.

Bhutta MS, Roy B, Gould GW, McInerny CJ - PLoS ONE (2014)

Synthetic septation phenotypes observed in double mutants in genes encoding ESCRT proteins and (a) plo1-ts35, (b) ark1-T8 or (c) clp1Δ.Fission yeast double mutant strains were grown in complete liquid medium at 25°C to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Scale bar, 10 µm. The frequency of phenotypes A–F (described in Figure 1a) was quantitatively analysed in strains containing double mutants, in comparison to each parent. In each case 400 cells were counted in triplicate (*p<0.05). Each of the ESCRT genes labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214795&req=5

pone-0111789-g002: Synthetic septation phenotypes observed in double mutants in genes encoding ESCRT proteins and (a) plo1-ts35, (b) ark1-T8 or (c) clp1Δ.Fission yeast double mutant strains were grown in complete liquid medium at 25°C to mid-exponential phase and harvested. Cells were stained with Calcofluor white and visualised using fluorescence microscopy. Both fluorescence and bright field images are shown. Scale bar, 10 µm. The frequency of phenotypes A–F (described in Figure 1a) was quantitatively analysed in strains containing double mutants, in comparison to each parent. In each case 400 cells were counted in triplicate (*p<0.05). Each of the ESCRT genes labels is accompanied by its respective ESCRT complex identification (E-0, E-I, E-II and E-III).
Mentions: To further understand the relationship between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase, double mutants were examined for potential septal defects. Double mutants were compared to both sets of respective mutant parents, and the septation profiles for plo1-ts35, clp1Δ, ark1-T8 and ark1-T11 single mutants relative to wild-type have been shown in Fig. S3. A higher proportion of phenotype Classes C–F was observed in plo1-ts35 cells, and of Classes C and F in clp1Δ cells (Fig. S3). Interestingly, plo1 mutants when combined with individual mutations in ESCRT genes frequently revealed a decreased proportion of the Class F phenotype, indicative of phenotypic rescue and the interaction of these genes within a shared pathway. For instance, the vps4Δ strain exhibited the Class F phenotype in 39% of cells; this was reduced to 23% in plo1-ts35 vps4Δ cells (p<0.05 relative to both parents; Fig. 2a). Both sets of double mutants also exhibited increases in phenotype Classes B–E (Fig. 2a and 2c). An increased proportion of Class B, E and F was observed in ark1-T8 cells (Fig. S3), and analysis of double mutants revealed a higher prevalence of Classes B in particular (Fig. 2b). A similar increase in Class B was observed in double mutants with ark1-T11 (Fig. S4). Collectively, these data are highly suggestive of genetic interactions between ESCRT and plo1, ark1 and cdc14 genes, and imply cooperation of these genes to control cell separation in this organism.

Bottom Line: Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic.Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase.Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
Cytokinesis and cell separation are critical events in the cell cycle. We show that Endosomal Sorting Complex Required for Transport (ESCRT) genes are required for cell separation in Schizosaccharomyces pombe. We identify genetic interactions between ESCRT proteins and polo and aurora kinases and Cdc14 phosphatase that manifest as impaired growth and exacerbated defects in septation, suggesting that the encoded proteins function together to control these processes. Furthermore, we observed defective endosomal sorting in mutants of plo1, ark1 and clp1, as has been reported for ESCRT mutants, consistent with a role for these kinases in the control of ESCRT function in membrane traffic. Multiple observations indicate functional interplay between polo and ESCRT components: firstly, two-hybrid in vivo interactions are reported between Plo1p and Sst4p, Vps28p, Vps25p, Vps20p and Vps32p; secondly, co-immunoprecipitation of human homologues of Vps20p, Vps32p, Vps24p and Vps2p by human Plk1; and thirdly, in vitro phosphorylation of budding yeast Vps32p and Vps20p by polo kinase. Two-hybrid analyses also identified interactions between Ark1p and Vps20p and Vps32p, and Clp1p and Vps28p. These experiments indicate a network of interactions between ESCRT proteins, plo1, ark1 and clp1 that coordinate membrane trafficking and cell separation in fission yeast.

Show MeSH