Profilin is required for the normal timing of actin polymerization in response to thermal stress

Abstract

We have used a fluorometric assay to determine the relative amounts of polymerized actin (F-actin) in wild-type and profilin mutant yeast cells. Our results indicate that profilin plays a role in maintaining normal F-actin levels in response to shifts to high temperature. Cells lacking prof'din display a greater drop in F-actin levels upon such temperature shifts, and are slower to recover to initial F-actin levels than are wild-type cells. Interestingly, shifts to cold temperatures result in rapid increases of F-actin levels in wild-type and profilin null cells. We have further determined that shifting to high-osmolarity growth conditions causes a relatively slow decrease in F-actin levels in wild-type cells, and a small but rapid increase in the F-actin levels in profilin null cells. Profilin null cells contain normal concentrations of F-actin while growing exponentially at room temperature, indicating that profilin is not essential for maintaining F-actin concentrations during steady-state growth. Our data suggest that actin is inherently unstable in vivo at high temperatures, and that profilin helps to maintain actin in its filamentous state at these temperatures, perhaps by stimulating actin polymerization in a proper temporal and spatial fashion. direct interaction between yeast profilin and adenylate cyclase (S. Palmieri and B. Haarer, unpublished data).Despite these many interactions attributed to profilins from various species, it is still unclear which roles are central to profilin function, and how such properties relate to the in vivo control of the actin cytoskeleton. Convincing arguments can be made for an in vivo role as an inhibitor [13][14][15], or stimulator [8,16] of actin polymerization. Indeed, it is entirely likely that profilin could be playing both roles, depending on species, cell type, or temporal regulation within an individual cell. To further address profilin's potential in vivo functions, we have utilized a fluorometric assay to measure the relative filamentous actin content in cells before and after changing growth conditions. Our findings are consistent with profilin's playing a significant role in the stimulation of actin polymerization under conditions that are perturbing to the yeast actin cytoskeleton. Materials and methods

The Inositol 5′-Phosphatase SHIP-2 Negatively Regulates IgE-Induced Mast Cell Degranulation and Cytokine Production

“…After two washes with HBSS, F-actin bound phalloidins were then eluted by incubating the cells with 500 l of 100% methanol for 2 h in the dark. Supernatants were collected and the amount of eluted phalloidins was measured by fluorometry (31).…”

Signal-Mediated Depolymerization of Actin in Pollen during the Self-Incompatibility Response

“…To investigate whether F-actin depolymerization was triggered by the SI response in pollen, we determined the amount of polymeric actin in poppy pollen using a biochemical method to quantify fluorescent phalloidin binding (Howard and Oresajo, 1985b;Lillie and Brown, 1994;Yeh and Haarer, 1996) that has been adapted for use in plant cells (Gibbon et al, 1999). Polymer levels in pollen during hydration, germination, and tube growth were measured ( Figure 2A).…”

“…In most signaling responses for which accurate measurements exist, extracellular stimuli lead to the polymerization of actin (Carlsson et al, 1979;Howard and Oresajo, 1985a;Hall et al, 1988;Greenberg et al, 1991;Downey et al, 1992;Becker and Hart, 1999). However, some examples of rapid actin depolymerization exist (Ding et al, 1991;Lillie and Brown, 1994;Yeh and Haarer, 1996;Aizawa et al, 2001). For example, during the heat shock response of yeast, a 25 to 50% reduction in F-actin is observed within 30 s, but levels return to normal within 3 min (Lillie and Brown, 1994).…”

Mutational and hyperexpression-induced disruption of bipolar budding in yeast

“…Profilin is an actinmonomer-binding protein that is important for vegetative growth and essential for normal polarized budding in yeast (Haarer et al, 1990 ;Sohn & Goldschmidt-Clermont, 1994). Deletion of PFY1 results in randomized budding in haploids (Haarer et al, 1990), while various PFY1 point mutations result in randomized budding in diploids, but do not affect the normal axial budding of haploid strains (Haarer et al, 1996 ;T. Freedman and B. Haarer, unpublished data).…”

“…Pseudohyphal growth also requires the actin cytoskeleton to properly organize in a polarized fashion (Cali et al, 1998). Indeed, mutations that perturb actin function can disrupt both bipolar budding and pseudohyphal growth, while having little or no adverse effect on axial budding (Haarer et al, 1996 ;Yang et al, 1997).…”