Abstract
SummaryActin 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.
Highlights
Live-cell analysis in budding yeast has led the way in understanding the role of actin during endocytosis
Imaging of fluorescently tagged reporters indicates that a similar sequential recruitment and disassembly of protein complexes occurs during mammalian endocytosis
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 [10]
Summary
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 [1,2,3,4]. While actin appears to serve an essential function when the plasma membrane is under tension [5], several studies indicate that actin is recruited to the majority of endocytic sites [6, 7]. 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 [8]. 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 [10]
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