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• W1970190717 abstract "•Yeast dynamin Vps1 forms oligomeric rings that bind and bundle F-actin in vitro•Mutations in the Vps1 stalk region disrupt actin binding and bundling•The Vps1 RR-EE actin binding mutant has defects in endocytic scission in vivo Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission. Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission. Live-cell analysis in budding yeast has led the way in understanding the role of actin during endocytosis. Actin is recruited and polymerized through the function of the nucleating Arp2/3 complex and its activator Las17/WASP at specific sites on the plasma membrane, most likely determined by concentrations of endocytic coat proteins and cargo [1Carroll S.Y. Stimpson H.E.M. Weinberg J. Toret C.P. Sun Y. Drubin D.G. Analysis of yeast endocytic site formation and maturation through a regulatory transition point.Mol. Biol. Cell. 2012; 23: 657-668Crossref PubMed Scopus (60) Google Scholar, 2Robertson A.S. Smythe E. Ayscough K.R. Functions of actin in endocytosis.Cell. Mol. Life Sci. 2009; 66: 2049-2065Crossref PubMed Scopus (97) Google Scholar, 3Urbanek A.N. Smith A.P. Allwood E.G. Booth W.I. Ayscough K.R. A novel actin-binding motif in Las17/WASP nucleates actin filaments independently of Arp2/3.Curr. Biol. 2013; 23: 196-203Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 4Weinberg J. Drubin D.G. Clathrin-mediated endocytosis in budding yeast.Trends Cell Biol. 2012; 22: 1-13Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar]. Growth of actin filaments by polymerization, and their movement by type 1 myosins (Myo3 and Myo5), then drives the formation of a pronounced invagination, around which further factors assemble to bring about constriction, scission, and release of the vesicle [2Robertson A.S. Smythe E. Ayscough K.R. Functions of actin in endocytosis.Cell. Mol. Life Sci. 2009; 66: 2049-2065Crossref PubMed Scopus (97) Google Scholar, 4Weinberg J. Drubin D.G. Clathrin-mediated endocytosis in budding yeast.Trends Cell Biol. 2012; 22: 1-13Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar]. Imaging of fluorescently tagged reporters indicates that a similar sequential recruitment and disassembly of protein complexes occurs during mammalian endocytosis. While actin appears to serve an essential function when the plasma membrane is under tension [5Boulant S. Kural C. Zeeh J.C. Ubelmann F. Kirchhausen T. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis.Nat. Cell Biol. 2011; 13: 1124-1131Crossref PubMed Scopus (379) Google Scholar], several studies indicate that actin is recruited to the majority of endocytic sites [6Merrifield C.J. Feldman M.E. Wan L. Almers W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.Nat. Cell Biol. 2002; 4: 691-698Crossref PubMed Scopus (561) Google Scholar, 7Taylor M.J. Perrais D. Merrifield C.J. A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.PLoS Biol. 2011; 9: e1000604Crossref PubMed Scopus (518) Google Scholar]. Recent evidence suggests a positive feedback loop functions at endocytic sites, with dynamin, actin, and N-BAR proteins co-operating to effect efficient scission of membranes to release a vesicle [8Taylor M.J. Lampe M. Merrifield C.J. A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.PLoS Biol. 2012; 10: e1001302Crossref PubMed Scopus (113) Google Scholar]. Quantitative fluorescence microscopy of dynamin-2 revealed that, while low levels of dynamin were present early in endocytosis, actin recruitment directly preceded the precise temporal recruitment of further dynamin, sufficient to generate a single dynamin ring at sites, prior to scission [9Grassart A. Cheng A.T. Hong S.H. Zhang F. Zenzer N. Feng Y. Briner D.M. Davis G.D. Malkov D. Drubin D.G. Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis.J. Cell Biol. 2014; 205: 721-735Crossref PubMed Scopus (142) Google Scholar]. While the interdependency of dynamin and actin is made clear by these studies, the mechanisms underlying these observations remains unknown. Another study has demonstrated a direct interaction between dynamin and actin, but while this was shown to be required for stress fiber formation in podocytes, mutations affecting dynamin actin interaction did not inhibit transferrin uptake suggesting that the direct interaction was not required for endocytosis [10Gu C. Yaddanapudi S. Weins A. Osborn T. Reiser J. Pollak M. Hartwig J. Sever S. Direct dynamin-actin interactions regulate the actin cytoskeleton.EMBO J. 2010; 29: 3593-3606Crossref PubMed Scopus (179) Google Scholar]. While the role of actin in driving membrane invagination in yeast is clear, its role in scission has not been closely studied. Scission function has been attributed to the amphiphysin dimer Rvs161/Rvs167 and to the dynamin homolog Vps1, with both proteins localizing to endocytic sites immediately prior to scission [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar]. The Rvs167 SH3 domain binds directly to Vps1, and in vivo a reduced level of amphiphysin is recruited to endocytic sites in the absence of vps1 [12Smaczynska-de Rooij I.I. Allwood E.G. Mishra R. Booth W.I. Aghamohammadzadeh S. Goldberg M.W. Ayscough K.R. Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.Traffic. 2012; 13: 317-328Crossref PubMed Scopus (39) Google Scholar]. When both components are deleted, >65% of invaginations show retraction back toward the plane of the plasma membrane indicative of a scission defect [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar]. Vps1 is one of three dynamin-like proteins in yeast but is the only one of these dynamins that functions in membrane trafficking pathways. Like other dynamins, Vps1 is predicted to have a GTPase domain and a stalk region. However, in common with non-classical dynamins it does not have a PH domain but an alternative domain referred to as Insert B. Despite the absence of the PH domain, Vps1 can bind and tubulate liposomes [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar]. The localization, interactions, and deletion phenotypes of Vps1 suggest that it is likely to function in a similar way to Dynamin-1 or Dynamin-2 in mammalian endocytosis [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar, 13Nannapaneni S. Wang D. Jain S. Schroeder B. Highfill C. Reustle L. Pittsley D. Maysent A. Moulder S. McDowell R. Kim K. The yeast dynamin-like protein Vps1:vps1 mutations perturb the internalization and the motility of endocytic vesicles and endosomes via disorganization of the actin cytoskeleton.Eur. J. Cell Biol. 2010; 89: 499-508Crossref PubMed Scopus (34) Google Scholar, 14Schmid S.L. Frolov V.A. Dynamin: Functional Design of a Membrane Fission Catalyst.Ann. Rev. Cell. Dev. Biol. 2011; 27Crossref PubMed Scopus (220) Google Scholar, 15Yu X. Cai M. The yeast dynamin-related GTPase Vps1p functions in the organization of the actin cytoskeleton via interaction with Sla1p.J. Cell Sci. 2004; 117: 3839-3853Crossref PubMed Scopus (53) Google Scholar]. In addition to a function in endocytosis, Vps1 also functions at other stages of membrane trafficking including Golgi to vacuole (lysosome) transport and endosomal recycling [16Bowers K. Stevens T.H. Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae.Biochim. Biophys. Acta. 2005; 1744: 438-454Crossref PubMed Scopus (237) Google Scholar, 17Briñas L. Vassilopoulos S. Bonne G. Guicheney P. Bitoun M. Role of dynamin 2 in the disassembly of focal adhesions.J. Mol. Med. 2013; 91: 803-809Crossref PubMed Scopus (7) Google Scholar, 18Durieux A.C. Vassilopoulos S. Lainé J. Fraysse B. Briñas L. Prudhon B. Castells J. Freyssenet D. Bonne G. Guicheney P. Bitoun M. A centronuclear myopathy—dynamin 2 mutation impairs autophagy in mice.Traffic. 2012; 13: 869-879Crossref PubMed Scopus (44) Google Scholar, 19González-Jamett A.M. Momboisse F. Haro-Acuña V. Bevilacqua J.A. Caviedes P. Cárdenas A.M. Dynamin-2 function and dysfunction along the secretory pathway.Front. Endocrinol. (Lausanne). 2013; 4: 126PubMed Google Scholar, 20Hoepfner D. van den Berg M. Philippsen P. Tabak H.F. Hettema E.H. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.J. Cell Biol. 2001; 155: 979-990Crossref PubMed Scopus (256) Google Scholar, 21Liu J. Kaksonen M. Drubin D.G. Oster G. Endocytic vesicle scission by lipid phase boundary forces.Proc. Natl. Acad. Sci. USA. 2006; 103: 10277-10282Crossref PubMed Scopus (147) Google Scholar, 22Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (678) Google Scholar, 23Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (735) Google Scholar]. Recently, Dynamin-2 has also been reported to function in a range of membrane trafficking events beyond endocytosis raising the possibility that dynamin function is more promiscuous than previously considered, and that dynamins may act as general scission factors on multiple membranes, possibly regulated by distinct components at each site [16Bowers K. Stevens T.H. Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae.Biochim. Biophys. Acta. 2005; 1744: 438-454Crossref PubMed Scopus (237) Google Scholar, 17Briñas L. Vassilopoulos S. Bonne G. Guicheney P. Bitoun M. Role of dynamin 2 in the disassembly of focal adhesions.J. Mol. Med. 2013; 91: 803-809Crossref PubMed Scopus (7) Google Scholar, 18Durieux A.C. Vassilopoulos S. Lainé J. Fraysse B. Briñas L. Prudhon B. Castells J. Freyssenet D. Bonne G. Guicheney P. Bitoun M. A centronuclear myopathy—dynamin 2 mutation impairs autophagy in mice.Traffic. 2012; 13: 869-879Crossref PubMed Scopus (44) Google Scholar, 19González-Jamett A.M. Momboisse F. Haro-Acuña V. Bevilacqua J.A. Caviedes P. Cárdenas A.M. Dynamin-2 function and dysfunction along the secretory pathway.Front. Endocrinol. (Lausanne). 2013; 4: 126PubMed Google Scholar, 20Hoepfner D. van den Berg M. Philippsen P. Tabak H.F. Hettema E.H. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.J. Cell Biol. 2001; 155: 979-990Crossref PubMed Scopus (256) Google Scholar, 21Liu J. Kaksonen M. Drubin D.G. Oster G. Endocytic vesicle scission by lipid phase boundary forces.Proc. Natl. Acad. Sci. USA. 2006; 103: 10277-10282Crossref PubMed Scopus (147) Google Scholar, 22Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (678) Google Scholar, 23Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (735) Google Scholar]. In this study, we have used both in vitro and in vivo approaches to address key questions about the functional interactions between actin and dynamin to gain mechanistic insight into the first membrane fission step of the endocytic pathway. A direct dynamin-actin interaction has previously only been reported for mammalian dynamin-1; thus, an initial step was to determine whether the interaction is conserved with yeast dynamin Vps1 and actin. Recombinant Vps1 was purified, incubated with F-actin, followed by centrifugation. Binding was assessed with both yeast actin (Figure 1A) and rabbit muscle actin (Figure S1A). An increase in amount of Vps1 associating with F-actin in the pellet was observed (Figure 1B; Figures S1B and S1E). The average KD of Vps1 from four independent experiments for yeast actin was calculated to be 1.9 ± 0.36 μM (KD for rabbit actin 0.92 ± 0.31 μM). Having demonstrated that Vps1 binds to actin, we aimed to determine whether mutations that compromise the Dynamin-1-actin interaction also affect the Vps1-actin interaction. Based on the crystal structure of dynamin, the suggested actin binding region lies along a helix that forms part of the central stalk domain [24Faelber K. Posor Y. Gao S. Held M. Roske Y. Schulze D. Haucke V. Noé F. Daumke O. Crystal structure of nucleotide-free dynamin.Nature. 2011; 477: 556-560Crossref PubMed Scopus (224) Google Scholar, 25Ford M.G.J. Jenni S. Nunnari J. The crystal structure of dynamin.Nature. 2011; 477: 561-566Crossref PubMed Scopus (206) Google Scholar]. Based on primary and secondary structure comparisons, this helix in Vps1 is well conserved making selection of charge-switch mutations straightforward (Figures 1C and 1D). All selected residues are predicted to project outward from the stalk region and were considered unlikely to disrupt secondary structure of the region. Mutants RR457,458EE (RR-EE), K453E R457E R458E (KRR-EEE), E461K, and E473K were generated both in plasmids and integrated into the yeast genome. Wild-type and mutant Vps1 were expressed, purified, and incubated with yeast F-actin and centrifuged to determine extent of binding (Figure 1E). Quantification of Vps1 pelleting (n ≥ 3 independent experiments) indicated that there was a significant reduction in actin binding of the RR-EE and the E473K mutants (p value from unpaired t test 0.009 for RR-EE; 0.0001 for E473K; Figure 1F). A similar pattern of binding occurred following incubation with rabbit muscle actin (data not shown). The mutations were predicted to not affect folding but to determine whether other functions of Vps1 were affected; lipid binding was also assessed. As we have shown previously, wild-type Vps1 is able to bind to liposomes [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar]. This binding interaction did not appear to be compromised in any of the Vps1 mutants (Figure S1C). Analysis of phenotypes arising from the mutations was then performed in vivo. The four mutants, a deletion, and a wild-type strain were tested for protein expression and overall growth phenotypes. All four mutants expressed in yeast with only the KRR-EEE mutant showing reduced levels compared to wild-type (Figure 2A). Deletion of vps1 causes a temperature-sensitive phenotype with cells unable to grow at 37°C [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar, 23Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (735) Google Scholar]. The mutants gave distinct phenotypes in growth assays with the KRR-EEE, RR-EE, and E473K mutations causing temperature sensitivity, while E461K-expressing cells rescued temperature sensitivity of the deletion strain. Sorbitol is able to rescue some temperature-sensitive phenotypes, especially those associated with actin defects in endocytosis [26Aghamohammadzadeh S. Ayscough K.R. Differential requirements for actin during yeast and mammalian endocytosis.Nat. Cell Biol. 2009; 11: 1039-1042Crossref PubMed Scopus (161) Google Scholar]. When sorbitol was present on plates at 37°C, the RR-EE mutant now showed growth, while the two other temperature-sensitive mutants showed no clear improvement in growth (Figure 2B). To determine whether the mutants affected some or all phenotypes associated with vps1 deletion, assays were performed including analysis of vacuole morphology (vps1Δ cells have a class F phenotype; with a single large vacuole surrounded by multiple small fragmented vacuoles, while wild-type cells usually contain two to five similarly sized vacuoles [22Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (678) Google Scholar]); endosomal recycling of SNARE reporter Snc1-GFP (mutants in endosomal recycling fail to show clear plasma membrane staining of the reporter [27Burston H.E. Maldonado-Báez L. Davey M. Montpetit B. Schluter C. Wendland B. Conibear E. Regulators of yeast endocytosis identified by systematic quantitative analysis.J. Cell Biol. 2009; 185: 1097-1110Crossref PubMed Scopus (89) Google Scholar]); and peroxisome fission in cells carrying a reporter GFP-PTS1 (vps1Δ have a single elongated peroxisome while wild-type cells have multiple small peroxisomes [20Hoepfner D. van den Berg M. Philippsen P. Tabak H.F. Hettema E.H. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.J. Cell Biol. 2001; 155: 979-990Crossref PubMed Scopus (256) Google Scholar]). In all these assays, two mutants, RR-EE and E461K, behaved similar to the wild-type VPS1 strain, while two mutants, KRR-EEE and E473K, behaved like the strain lacking vps1 (Figure 2C). Carboxypeptidase Y, an enzyme that is cleaved to its mature form (mCPY) in the vacuole, is less efficiently processed in the vps1 deletion and a precursor accumulates (pCPY) [22Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (678) Google Scholar, 23Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (735) Google Scholar]. As shown (Figure 2D), pCPY accumulates in the KRR-EEE and E473K mutants, indicating that they share this trafficking defect with the vps1 deletion strain. Finally, analysis of lucifer yellow (LY) uptake was performed to assess fluid phase endocytosis (Figures 2E and 2F). Wild-type cells internalize LY by endocytosis and traffic it to the vacuole, while vps1 deletion cells accumulate the dye in vesicles and endosomes. Interestingly, RR-EE and E461K mutants that behaved similarly to wild-type cells in other assays, showed defects in their ability to traffic LY to the vacuole. Analysis of the proportion of cells with LY predominantly at the plasma membrane, endosomes, or vacuole at 60 min is shown in Figure S2. Together, these data indicate that KRR-EEE and E473K mutants behave largely as non-functional proteins with all in vivo phenotypes indistinguishable from cells completely lacking Vps1. The Vps1 E461K mutant does not significantly affect actin binding and behaves like wild-type Vps1 in most cell assays, with just a delay in endocytosis. In contrast, the mutation RR457-458EE revealed a specific endocytosis defect in vivo and reduced actin binding in vitro. A more detailed analysis of endocytic defects in the vps1 RR-EE mutant would allow us to understand the functional links between actin binding and endocytosis more clearly. However, in order to corroborate the data from the F-actin binding assays (Figures 1E and 1F), it was important to first substantiate the in vitro result with affinity measurements. Therefore, further binding assays were undertaken with the vps1 RR-EE mutant to measure the actin binding affinity. This analysis revealed an affinity of vps1 RR-EE for yeast actin, with a KD of 4.8 ± 2.6 μM (compared to 1.9 μM for wild-type), supporting the previous data that this mutant has a reduced binding capacity for actin (Figure S1D). Given that functions other than endocytosis were rescued in cells expressing the vps1 RR-EE mutant, it was first important to establish whether this mutant protein is able to localize to sites of endocytosis or whether endocytosis was defective due to lack of Vps1 recruitment to these sites. Two approaches were used to demonstrate cortical patch localization. First, colocalization of Vps1-GFP (WT or mutant protein) with the reporter protein Abp1 was analyzed using TIRF microscopy. As shown in Figure 3A, clear puncta could be seen for both WT and mutant Vps1 proteins, and these showed co-localization in a temporal manner with Abp1 at the cell cortex (% co-localization wild-type 24% ± 4.1% SEM n = 8 cells; vps1 RR-EE 21.5% ± 5.1% SEM, n = 7). This relatively low co-localization is similar to that observed previously and probably reflects difficulties with incorporation of GFP tagged Vps1 into functionally relevant structures (see later analysis). Second a bimolecular fluorescence complementation (BiFC) approach was used in which each half of the yellow fluorescent protein variant Venus was appended to either Vps1 or the RR-EE mutant (VN) or to the amphiphysin Rvs167, which functions at endocytic sites but not on endosomes (VC). As shown (Figure S3), Rvs167-VC and Vps1-VN show clear punctate sites of interaction at the cell cortex in an organization indicative of endocytic sites. Again, the Vps1 RR-EE mutant exhibited similar localization to the wild-type protein. Reporter proteins that assemble at the endocytic site have been widely used to determine whether mutations or deletions cause defects at distinct stages of endocytosis [28Kaksonen M. Sun Y. Drubin D.G. A pathway for association of receptors, adaptors, and actin during endocytic internalization.Cell. 2003; 115: 475-487Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 29Kaksonen M. Toret C.P. Drubin D.G. A modular design for the clathrin- and actin-mediated endocytosis machinery.Cell. 2005; 123: 305-320Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar]. Previously, we demonstrated that deletion of vps1 caused a defect in both invagination and scission events [11Smaczynska-de Rooij I.I. Allwood E.G. Aghamohammadzadeh S. Hettema E.H. Goldberg M.W. Ayscough K.R. A role for the dynamin-like protein Vps1 during endocytosis in yeast.J. Cell Sci. 2010; 123: 3496-3506Crossref PubMed Scopus (82) Google Scholar, 12Smaczynska-de Rooij I.I. Allwood E.G. Mishra R. Booth W.I. Aghamohammadzadeh S. Goldberg M.W. Ayscough K.R. Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.Traffic. 2012; 13: 317-328Crossref PubMed Scopus (39) Google Scholar]. Analysis of proteins Sla1-GFP, Abp1-mCherry, Sla2-GFP, and Rvs167-GFP was used to gain further insights to the stage of endocytosis that is defective in the vps1 RR-EE mutant. Kymograph analysis of co-expressed Sla1-GFP and Abp1-mCherry indicated that the endocytic coat assembled appropriately in cells but that there were defects in invagination and scission in the vps1-null and mutant strain. Due to the highly aberrant behaviors observed, timing of patch lifetimes was considered unreliable, and instead the behavior of individual patches was categorized as normal invagination; no/short invagination; or aberrant scission. This latter class included patches that appeared to invaginate but then either retract back toward the plasma membrane (retraction) and those that remained invaginated for several seconds before reporters disassembled (delayed scission). Examples of these behaviors as patch tracks are shown on kymographs and patch tracks (Figures 3B and 3C). A summary graph of the Abp1-mCherry patch behaviors is shown (Figure 3D). Both the vps1-null and the vps1 RR-EE-expressing strain show fewer normal invaginations compared to those in wild-type cells (in multiple t test p ≤ 0.0001). Interestingly, the vps1 RR-EE mutant showed a higher proportion of aberrant scission than the vps1-null strain (p ≤ 0.0002 in t test), while the null strain showed increases in both invagination and scission defects. The behavior of an endocytic coat protein Sla2 was also analyzed in cells expressing wild-type and mutant Vps1 (Figure 3E for kymographs). Again, the mutant appeared to show a distinct phenotype, with patches in the RR-EE mutant mostly showing invagination from the plane of the membrane followed by a retraction or other aberrant movement before disassembly. A summary graph of Sla2-GFP patch behavior is shown (Figure 3F). As with Abp1, the proportion of scission defects is greater in the mutant compared to the null strain (p value ≤ 0.0001 in t test). Previously, we demonstrated that deletion of vps1 led to a reduction in the Rvs167 signal at the endocytic site suggesting that Vps1 might play a role in stabilizing Rvs167 at sites or might facilitate its oligomerization [12Smaczynska-de Rooij I.I. Allwood E.G. Mishra R. Booth W.I. Aghamohammadzadeh S. Goldberg M.W. Ayscough K.R. Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.Traffic. 2012; 13: 317-328Crossref PubMed Scopus (39) Google Scholar]. The lifetime and intensity of the Rvs167 signal in the vps1 mutant was analyzed and compared to VPS1 wild-type and null strains. As shown in Figures 3G and 3H, the vps1 RR-EE mutant shows no significant differences from wild-type in intensity or lifetime of Rvs167-GFP staining, indicating that amphiphysin can be recruited and maintained correctly in the mutant strain. While observation of retracting endocytic patches is indicative of a scission problem, clearer insight into the defect caused by vps1 RR-EE mutation used electron microscopy to analyze invaginations. As shown (Figure 4), this approach revealed a dramatic increase in the length of invaginations observed in cells expressin" @default.
• W1970190717 created "2016-06-24" @default.
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• W1970190717 date "2015-03-01" @default.
• W1970190717 modified "2023-10-10" @default.
• W1970190717 title "A Dynamin-Actin Interaction Is Required for Vesicle Scission during Endocytosis in Yeast" @default.
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