Using total internal reflection microscopy (TIRFM), we recently performed an extensive analysis of actin cable dynamics in S. cerevisiae
We found that cables were reorganized throughout the cell cycle via a combination of growth, shrinkage, bending, bundling and translational motility.16,17
In contrast to previously published results,14,15
cable extension and movement occurred at a wide range of speeds between 0.5 and 5 µm/s. To our surprise, cable velocity and turnover were highest in unpolarized G1
cells, and reduced upon cell polarization. To determine the molecular basis for cable dynamics we analyzed cable dynamics in various mutants of actin regulators and polarisome components. We found that the formin Bni1 was associated with cable extension at 1–2 µm/s, while the other formin Bnr1 mediated slower cable extension at rates below 1 µm/s. In addition, we could show that the type V myosin Myo2 drove rapid translational movement of cables along the cell periphery at speeds above 2 µm/s.
The contribution of formins and Myo2 were differentially regulated through the cell cycle. In unpolarized G1
cells, cable dynamics was dominated by Bni1 and Myo2. Strikingly, both molecules assembled into distinct cortical patches associated with the plasma membrane. Double color TIRFM experiments suggested that Bni1 patches were sites of actin cable assembly, whereas Myo2 patches generated translational motility by sliding actin cables along the inner plasma membrane surface. Upon cell polarization, cortical Bni1 and Myo2 patches in mother cells became destabilized and both proteins now instead became concentrated at polarization sites and bud tips. At the same time Bnr1 was activated and localized to the bud neck. As a consequence of these changes, Myo2 no longer participated in cable motility. Bnr1, with an actin binding affinity tenfold higher than Bni1,18
dominated cable dynamics in mother cells, resulting in the observed slow-down of cable reorganization in polarized cells.
Our findings can be summarized in a model of dynamic actin reorganization through spatio-temporal interplay of three motor molecules with different modes of action and kinetics (reviewed in ref. 16
, ). It was shown previously that the two formins Bni1 and Bnr1 have distinct biochemical properties and localize to different cellular locations.9,18
We now demonstrated that in cells these differences are actually used to generate kinetically distinct actin cables. Combined with the stable association of Bnr1 with septins at the bud neck19
and the dynamic localization of Bni1 on the mother cell cortex,9
this enables cells to switch between two different modes of actin organization by simple activation/inactivation of the formin Bnr1.
Figure 1 Proposed mechanism of actin cable dynamics driven by formin and Myosin V. (A) Actin cables are generated by cortical Bni1 molecules. (B) Actin cables can serve as tracks for Myo2 to drive cargo transport. (C) Cortically anchored Myo2 can function as a (more ...)