Tropomyosin and Profilin Cooperate to Promote Formin-Mediated Actin Nucleation and Drive Yeast Actin Cable Assembly

Abstract

Tropomyosins comprise a large family of actin-binding proteins with critical roles in diverse actin-based processes [1], but our understanding of how they mechanistically contribute to actin filament dynamics has been limited. We addressed this question in S. cerevisiae, where tropomyosins (Tpm1 and Tpm2), profilin (Pfy1), and formins (Bni1 and Bnr1) are required for the assembly of an array of actin cables that facilitate polarized vesicle delivery and daughter cell growth. Formins drive cable formation by promoting actin nucleation and by accelerating actin filament elongation together with profilin [2]. In contrast, how tropomyosins contribute mechanistically to cable formation has been unclear, but genetic studies demonstrate that Tpm1 plays a more important role than Tpm2 [3, 4]. Here, we found that loss of TPM1 in strains lacking BNR1, but not BNI1, leads to severe defects in cable formation, polarized secretion, and cell growth, suggesting that TPM1 function is required for proper Bni1-mediated cable assembly. Furthermore, in vitro total internal reflection fluorescence (TIRF) microscopy demonstrated that Tpm1 strongly enhances Bni1-mediated, but not Bnr1-mediated, actin nucleation without affecting filament elongation rate, whereas Tpm2 has no effects on Bni1 or Bnr1. Tpm1 stimulation of Bni1-mediated nucleation also requires profilin and its interactions with both G-actin and formins. Together, these results demonstrate that yeast Tpm1 works in concert with profilin to promote formin-dependent nucleation of actin cables, thus expanding our understanding of how specific tropomyosin isoforms influence actin dynamics.

Keywords: actin; formin; profilin; tropomyosin; yeast.

Figure 1. Tpm1 is critical for Bni1-dependent actin cable formation and cell growth

(A) Five-fold serial dilutions of yeast strains grown on YEPD plates at indicated temperatures. (B) Growth curves for indicated yeast strains grown in YEPD at 34°C with shaking in a microplate absorbance reader with OD₆₀₀ measured every 5 min. Data averaged from ≥18 replicates/strain. Lighter shading, SEM. (C) Representative DIC images of cells grown to mid-log phase at 25°C in Y EPD and fixed. Scale bar, 5 μm. (D) Representative images of small, medium, and large budded cells grown at 25°C in YEPD, fixed, and stained with Alexafluor-488 phalloidin. Scale bar, 3 μm. (E) Average number of actin cables visible in the mother compartment (n ≥ 50 cells analyzed per strain). Student’s T-test used to determine significant difference (p < 0.05) from wildtype (‘w’), appropriate tropomyosin single mutant (‘t’), or appropriate formin single mutant (‘f’). See also Figure S1.

Figure 2. Efficient secretory vesicle traffic requires Tpm1 function in Bni1-mediated actin cable assembly

(A) Representative images of early log phase cells showing distribution of GFP-Sec4 (false colored). Scale bar, 3 μm. (B) Ratio of bud to mother GFP-Sec4 fluorescence. Each dot is the ratio quantified for one cell (n ≥ 40 cells per strain analyzed). Black line, Mean. Student’s T-test used to determine the indicated significant difference (p < 0.05) from wildtype (‘w’), appropriate tropomyosin single mutant (‘t’), or appropriate formin single mutant (‘f’). (C) Ratio of bud to mother GFP-Sec4 fluorescence plotted as a function of bud size (same data as in B). Lines fit by linear regression using standard equations in Prism 6.

Figure 3. Tpm1 and Tpm2 effects on formin-mediated actin filament nucleation and elongation

(A) Actin nucleation effects determined by in vitro TIRF microscopy. Reactions contained 1 μM monomeric actin (20% Oregon Green-labeled), 3 μM profilin, and variable components: 250 pM Bni1, 200 pM Bnr1, 2.5 μM am-Tpm1, and 2.5 μM am-Tpm2. Number of filaments per field of view (FOV) was quantified 5 min after initiation of actin assembly from four independent experiments (16 FOV per condition). (B) Actin filament elongation rates determined from the same TIRF reactions as A. Elongation rates were calculated from the slopes of individual filament traces of length versus time (n ≥ 30 filaments per condition). (C) Representative time-lapse images from TIRF reactions. Scale bar, 20 μm. Error bars, SEM. Student’s T-test used to determine significant differences: n.s. – not significant, * < 0.05, ** < 0.01, *** < 0.001. See also Figure S2 and Movies S1 & S2.

Figure 4. Tpm1 stimulation of Bni1-mediated actin nucleation requires profilin and its interactions with G-actin and formins

(A) Representative FOVs from TIRF reactions containing 1.0 μM monomeric actin (20% Oregon Green-labeled) polymerized in the presence of 250 pM Bni1 and variable components: 3.0 μM yeast profilin and 2.5 μM am-Tpm1. Scale bar, 20 μm. (B) Actin nucleation effects determined from the reactions in A. Number of filaments per FOV was quantified as in Figure 3A from two independent experiments (12 FOV per condition). (C) Representative FOVs from TIRF reactions; conditions as in A, except using 3 μM Pfy1 and Pfy1-19 as indicated. Scale bar, 20 μm. (D) Actin nucleation effects determined by TIRF microscopy; conditions as in A, except using 3 μM Pfy1, Pfy1-4, or Pfy1-19 as indicated. The number of filaments per FOV was quantified as in Figure 3A from ≥ 2 separate experiments (≥ 12 FOVs per condition). (E) Native gel shift analysis of G-actin. Reactions contained 1.0 μM latrunculin bound G-actin with one or more variable components: 0.5 μM Bni1, 5.0 μM am-Tpm1, and 3 μM yeast profilin (Pfy1). Reactions fractionated by native PAGE, stained with Coomassie Blue and actin band intensity was quantified at the unshifted (blue arrow) and shifted (red arrow) positions. Data averaged from three independent experiments (blue and red bars, as above). Significant differences are compared to actin & profilin control (Lane 2). (F) Two possible models for how Tpm1 and profilin works together to enhance Bni1-mediated actin filament nucleation. (G) Graphical summary of actin cable pheontypes in indicated strains studied. Error bars, SEM. Student’s T-test used to determine significant differences: n.s. – not significant, * < 0.05, ** < 0.01, **** < 0.0001. See also Figure S3 and Movies S3, S4, and S5.