Novel Role of the Endocytic Adaptor Epsin-2 in Cytokinesis Regulation.

DEBARATI MUKHERJEE1, BRIAN G. COON1, SILVIA A. LONGHI2, 3, BEVERLY WENDLAND2, LILIA A. RETEGUI AND R. CLAUDIO AGUILAR

VILLARS-SUR-OLLON - SWITZERLAND

Workshop; WORKSHOP ON ENDOCYTIC SYSTEMS: MECHANISM AND FUNCTION; 2007

EMBO -FEBS

The Epsins are a family of adaptors involved in the recruitment of the endocytic machinery, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic lipid-binding Epsin N-Terminal Homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. Recently we reported that, besides their classical endocytic function, Epsins regulate the activation of the small GTPase Cdc42. Here we present results from genetic, cell biological and biochemical studies performed on Saccharomyces cerevisiae that expand the repertoire of Epsins�� functions to cytokinesis regulation. Thus, overexpression of the ENTH domain of Epsin-2 (ENTH2), but not Epsin-1, promotes dramatic abnormalities in cytokinesis. Specifically, cells with high ENTH2 dosage generate chains of multiple buds and exhibit massive defects in Septin assembly. Genetic and cell biological evidence demonstrates that this dominant negative effect of ENTH2 leads to aberrant regulation of the Swe1-dependent morphogenesis checkpoint without affecting cell-cycle progression. Our results also implicate another Ras-related small GTPase: Rsr1, which is involved in bud site selection, pseudo-hyphal growth and is an upstream regulator of Cdc42. GFP-Epsin-2 localizes at sites of bud emergence and the bud-neck region supporting the idea of a physiological role for Epsin-2 at early stages of cell division. At a molecular level, we analyzed structural models taking into consideration criteria such as solvent accessibility and relative conservation of residues in ENTH1 vs. ENTH2 and identified the specific ENTH2 region responsible for induction of the cytokinesis defects. Importantly, overexpression of ENTH2 in HeLa cells also leads to cytokinesis arrest, suggesting that the pathway affected by ENTH2 is conserved in mammals. Collectively, our novel findings provide a potential molecular mechanism linking endocytosis (via Epsin-2) with signaling pathways regulating cytokinesis.via Epsin-2) with signaling pathways regulating cytokinesis.that expand the repertoire of Epsins�� functions to cytokinesis regulation. Thus, overexpression of the ENTH domain of Epsin-2 (ENTH2), but not Epsin-1, promotes dramatic abnormalities in cytokinesis. Specifically, cells with high ENTH2 dosage generate chains of multiple buds and exhibit massive defects in Septin assembly. Genetic and cell biological evidence demonstrates that this dominant negative effect of ENTH2 leads to aberrant regulation of the Swe1-dependent morphogenesis checkpoint without affecting cell-cycle progression. Our results also implicate another Ras-related small GTPase: Rsr1, which is involved in bud site selection, pseudo-hyphal growth and is an upstream regulator of Cdc42. GFP-Epsin-2 localizes at sites of bud emergence and the bud-neck region supporting the idea of a physiological role for Epsin-2 at early stages of cell division. At a molecular level, we analyzed structural models taking into consideration criteria such as solvent accessibility and relative conservation of residues in ENTH1 vs. ENTH2 and identified the specific ENTH2 region responsible for induction of the cytokinesis defects. Importantly, overexpression of ENTH2 in HeLa cells also leads to cytokinesis arrest, suggesting that the pathway affected by ENTH2 is conserved in mammals. Collectively, our novel findings provide a potential molecular mechanism linking endocytosis (via Epsin-2) with signaling pathways regulating cytokinesis.via Epsin-2) with signaling pathways regulating cytokinesis.overexpression of the ENTH domain of Epsin-2 (ENTH2), but not Epsin-1, promotes dramatic abnormalities in cytokinesis. Specifically, cells with high ENTH2 dosage generate chains of multiple buds and exhibit massive defects in Septin assembly. Genetic and cell biological evidence demonstrates that this dominant negative effect of ENTH2 leads to aberrant regulation of the Swe1-dependent morphogenesis checkpoint without affecting cell-cycle progression. Our results also implicate another Ras-related small GTPase: Rsr1, which is involved in bud site selection, pseudo-hyphal growth and is an upstream regulator of Cdc42. GFP-Epsin-2 localizes at sites of bud emergence and the bud-neck region supporting the idea of a physiological role for Epsin-2 at early stages of cell division. At a molecular level, we analyzed structural models taking into consideration criteria such as solvent accessibility and relative conservation of residues in ENTH1 vs. ENTH2 and identified the specific ENTH2 region responsible for induction of the cytokinesis defects. Importantly, overexpression of ENTH2 in HeLa cells also leads to cytokinesis arrest, suggesting that the pathway affected by ENTH2 is conserved in mammals. Collectively, our novel findings provide a potential molecular mechanism linking endocytosis (via Epsin-2) with signaling pathways regulating cytokinesis.via Epsin-2) with signaling pathways regulating cytokinesis.. Specifically, cells with high ENTH2 dosage generate chains of multiple buds and exhibit massive defects in Septin assembly. Genetic and cell biological evidence demonstrates that this dominant negative effect of ENTH2 leads to aberrant regulation of the Swe1-dependent morphogenesis checkpoint without affecting cell-cycle progression. Our results also implicate another Ras-related small GTPase: Rsr1, which is involved in bud site selection, pseudo-hyphal growth and is an upstream regulator of Cdc42. GFP-Epsin-2 localizes at sites of bud emergence and the bud-neck region supporting the idea of a physiological role for Epsin-2 at early stages of cell division. At a molecular level, we analyzed structural models taking into consideration criteria such as solvent accessibility and relative conservation of residues in ENTH1 vs. ENTH2 and identified the specific ENTH2 region responsible for induction of the cytokinesis defects. Importantly, overexpression of ENTH2 in HeLa cells also leads to cytokinesis arrest, suggesting that the pathway affected by ENTH2 is conserved in mammals. Collectively, our novel findings provide a potential molecular mechanism linking endocytosis (via Epsin-2) with signaling pathways regulating cytokinesis.via Epsin-2) with signaling pathways regulating cytokinesis.our novel findings provide a potential molecular mechanism linking endocytosis (via Epsin-2) with signaling pathways regulating cytokinesis.via Epsin-2) with signaling pathways regulating cytokinesis.