Abstract: Cortical actin patches are dynamic structures required for endocytosis in yeast [1Engqvist-Goldstein A.E. Drubin D.G. Actin assembly and endocytosis from yeast to mammals.Annu. Rev. Cell Dev. Biol. 2003; 19: 287-332Crossref PubMed Scopus (478) Google Scholar]. Recent studies have shown that components of cortical patches localize to the plasma membrane in a precisely orchestrated manner, and their movements at and away from the plasma membrane may define the endocytic membrane invagination and vesicle scission events, respectively [2Kaksonen 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 (507) Google Scholar]. Here, through live-cell imaging, we analyze the dynamics of the highly conserved class I unconventional myosin, Myo5, which also localizes to cortical patches and is known to be involved in endocytosis and actin nucleation [3Goodson H.V. Anderson B.L. Warrick H.M. Pon L.A. Spudich J.A. Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5) myosin I proteins are required for polarization of the actin cytoskeleton.J. Cell Biol. 1996; 133: 1277-1291Crossref PubMed Scopus (182) Google Scholar, 4Anderson B.L. Boldogh I. Evangelista M. Boone C. Greene L.A. Pon L.A. The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization.J. Cell Biol. 1998; 141: 1357-1370Crossref PubMed Scopus (101) Google Scholar, 5Geli M.I. Riezman H. Role of type I myosins in receptor-mediated endocytosis in yeast.Science. 1996; 272: 533-535Crossref PubMed Scopus (227) Google Scholar, 6Lechler T. Shevchenko A. Li R. Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization.J. Cell Biol. 2000; 148: 363-373Crossref PubMed Scopus (171) Google Scholar, 7Lechler T. Jonsdottir G.A. Klee S.K. Pellman D. Li R. A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.J. Cell Biol. 2001; 155: 261-270Crossref PubMed Scopus (92) Google Scholar, 8Evangelista M. Klebl B.M. Tong A.H. Webb B.A. Leeuw T. Leberer E. Whiteway M. Thomas D.Y. Boone C. A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex.J. Cell Biol. 2000; 148: 353-362Crossref PubMed Scopus (188) Google Scholar]. Myo5 exhibits a pattern of dynamic localization different from all cortical patch components analyzed to date. Myo5 associates with cortical patches only transiently and remains stationary during its brief cortical lifespan. The peak of Myo5 association with cortical patches immediately precedes the fast movement of Arp2/3 complex-associated structures away from the plasma membrane, thus correlating precisely with the proposed vesicle scission event. To further test the role of Myo5, we generated a temperature-sensitive mutant myo5 allele. In the myo5 mutant cells, Myo5 exhibits a significantly extended cortical lifespan as a result of a general impairment of Myo5 function, and Arp2 patches exhibit an extended slow-movement phase prior to the fast movement toward the cell interior. The myo5 mutant cells are defective in fluid-phase endocytosis and exhibit an increased number of invaginations on the membrane. Based on these results, we hypothesize that the myosin I motor protein facilitates the membrane fusion/vesicle scission event of endocytosis.