2011 Theses Doctoral

Actin Cable Function and Regulation in the Budding Yeast, Saccharomyces cerevisiae

Lipkin, Thomas Gregory Karney

In the following chapters, I describe factors underlying actin cable dynamics and assembly in the budding yeast, S. cerevisiae. First, I examined the role of type II myosin and a tropomyosin isoform in retrograde actin flow (Chapter II). In yeast and other cell types, actin undergoes retrograde or centripetal movement from the cell cortex towards the interior of the cell. Retrograde actin flow drives intracellular and cellular movement. Previous work in the Pon laboratory showed that actin cables undergo retrograde flow, which occurs, in part, from the force generated from actin polymerization and assembly at the elongating filament end. First, we find that the type II myosin, Myo1p, facilitates retrograde flow. We found that the rate of retrograde actin cable flow is reduced by 1) deletion of Myo1p, 2) displacement of Myo1p from the bud neck, or 3) a conditional mutation that inhibits Myo1p motor activity. These findings indicate that myosin motor activity provides the pulling force to drive movement of elongating actin cables from their site of assembly in the bud tip toward the mother cell. Additional work found that a tropomyosin isoform, Tpm2p, negatively regulates retrograde flow through inhibition of type II myosin binding to F-actin within actin cables. Since type II myosins and tropomyosins have a similar function in retrograde actin flow in animals cells, these findings provide the first evidence that yeast can be used as a model system to study this fundamental, conserved mechanism for actin dynamics. Second, I conducted a drug-based screen for novel regulators of actin cables (Chapter III).

Previous studies revealed a role for the yeast formins (Bni1p and Bnr1p) in stimulating polymerization of F-actin for actin cable formation, elongation and retrograde flow, and for other actin cable constituents including tropomyosins and actin bundling proteins in stabilizing and organizing F-actin within actin cables. Earlier work has revealed both that actin cables are selectively destabilized by low levels of the actin-destabilizing drug Latrunculin-A (Lat-A), and this drug inhibits cell growth. I carried out a screen designed to identify non-essential gene deletions that reduce the sensitivity of yeast to the growth inhibiting effects of low doses of Lat-A. Eighteen out of 4,848 deletion strains comprising the yeast deletion library exhibited reduced sensitivity to low levels of Lat-A. Eight of the genes represent uncharacterized open reading frames (ORFs) or encode proteins with no known function or activity. Deletion of a majority of these gene results in increased actin cable number. Additionally, I found the growth inhibiting effects of Lat-A are not suppressed by 1) overexpression of either of TPM1 or TPM2 or 2) deletion of TPM2 and the associated increase in the rate of retrograde actin cable flow.

Moreover, I found that one of the genes that reduces the growth-inhibiting effects of Lat-A, YHR022c, is an uncharacterized ORF which encodes a novel Ras-like protein. We call this gene Rar1p for Ras-like actin cable regulator. I found that deletion of RAR1 or expression of a constitutively active formin (Bni1p) produces similar phenotypes: 1) increased actin cable content in the presence and absence of low levels of Lat-A, 2) increased retrograde actin cable flow rates, and 3) resistance to Lat-A-dependent inhibition of growth. Finally, I found that the increase in actin cable content observed upon deletion of RAR1 requires Bni1p and not Bnr1p. Our findings reveal a role for previously uncharacterized genes in the regulation of actin cable stability, and new roles for previously characterized, conserved genes in this process. Equally important, I identified a novel Ras-like protein, Rar1p, and found that it affects actin cable abundance and sensitivity to Lat-A by functioning as an isoform-specific, negative regulator of the formin protein Bni1p. Chapter IV describes future directions for the work outlined in chapters II and III.