Studies of budding yeast, fission yeast, and animal cells identified many proteins that assemble and disassemble at endocytic sites. These observations support our two-zone hypothesis to explain endocytic tubule elongation and vesicle scission in fission yeast.Ĭlathrin-mediated endocytosis recycles membrane receptors and takes up nutrients. Cells lacking either nucleation-promoting factor assemble only one, stationary, zone of actin filaments. Wsp1p appears simultaneously at this site but subsequently moves away from the cell surface as it stimulates Arp2/3 complex to assemble a second zone of actin filaments. Myo1p concentrates at the site of endocytosis and initiates a zone of actin filaments assembled by Arp2/3 complex. The nucleation promoting factors Wsp1p (WASp) and Myo1p (myosin-I) define two independent pathways that recruit Arp2/3 complex, which assembles two zones of actin filaments. We used high-speed superresolution localization microscopy of live fission yeast cells to improve the spatial resolution to ∼35 nm with 1-s temporal resolution. The molecular mechanism by which actin filament polymerization deforms the cell membrane is unknown, largely due to lack of knowledge about the organization of the regulatory proteins and actin filaments. To internalize nutrients and cell surface receptors via clathrin-mediated endocytosis, cells assemble at least 50 proteins, including clathrin, clathrin-interacting proteins, actin filaments, and actin binding proteins, in a highly ordered and regulated manner.

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