Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik1 recruits approximately equal amounts of Pac1 and Dyn1. Overexpression of Pac1 enhanced plus end targeting of Dyn1 and vice versa, while affinity-purification of Dyn1 revealed that it exists in a complex with Pac1 in the absence of Bik1, leading us to conclude that the Pac1-Dyn1 complex preassembles in the cytoplasm prior to loading onto Bik1-decorated plus ends. Strikingly, we found that Pac1-overexpression augments cortical dynein activity through a mechanism distinct from loss of She1, a negative regulator of dynein-dynactin association. While Pac1-overexpression enhances the frequency of cortical targeting for dynein and dynactin, the stoichiometry of these complexes remains relatively unchanged at the plus ends compared to that in wild-type cells (~3 dynein to 1 dynactin). Loss of She1, however, enhances dynein-dynactin association at the plus ends and the cell cortex, resulting in an apparent 1:1 stoichiometry. Our results reveal differential regulation of cortical dynein activity by She1 and Pac1, and provide a potentially new regulatory step in the off-loading model for dynein function.

SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling.

The Asterless N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas its C terminus stabilizes Plk4 during mitosis.