Fimbrin and tropomyosin competition regulates endocytosis and cytokinesis kinetics in fission yeast

Abstract

Background: Tropomyosin is an important actin filament-stabilizing protein that controls the access of other essential proteins to filaments, including myosin motors, Arp2/3 complex, formin, and cofilin. It is therefore critical to establish mechanisms for regulating the actin filament binding of tropomyosin. We examined how the actin filament crosslinking protein fimbrin Fim1p and tropomyosin Cdc8p affect each other's ability to bind filaments, localize to particular cellular structures, and regulate filament severing by cofilin Adf1p in fission yeast Schizosaccharomyces pombe.

Results: We discovered a novel mechanism for regulating actin filament dynamics in fission yeast. Fim1p inhibits Cdc8p binding to actin filaments in vitro, which permits Adf1p-mediated severing in the presence of Cdc8p. In cells, the balance between Fim1p and Cdc8p is important for both endocytic actin patch kinetics and contractile ring assembly during cytokinesis. High Fim1p concentrations prevent Cdc8p from associating with actin patches, allowing rapid patch turnover and motility. In the absence of Fim1p, ectopic localization of Cdc8p to actin patches increases patch lifetime while decreasing patch motility. Fim1p and Cdc8p also play antagonistic roles during cytokinesis, in which the deletion of Fim1p rescues the contractile ring assembly defects caused by mutation of Cdc8p.

Conclusion: Fimbrin Fim1p dissociates tropomyosin Cdc8p from actin filaments, permitting cofilin Adf1p-mediated severing. Therefore, we propose that in addition to actin filament crosslinking, Fim1p has a novel role as a positive actin-binding "selector" protein that promotes the access of other proteins to actin filaments by inhibiting Cdc8p.

Figure 1

Fim1p prevents Cdc8p from binding actin filaments in vitro (A-D) Sedimentation of F-actin assembled from 3.0 μM Mg-ATP-actin monomers. (A and C) Coomassie Blue-stained gels of pellets after a range of Fim1p concentrations were added to filaments pre-incubated with 2.0 μM Cdc8p, and then spun at high speed 100,000g (A) or low speed 10,000g (C). Fim1p, actin and Cdc8p are marked on the right; molecular weights are indicated on the left. (B) Plot of the amount of Cdc8p (closed symbols) or Fim1p (open symbols) in the high speed pellet over a range of Fim1p concentrations. Results are shown for different orders of addition: Fim1p, Cdc8p, spin (●,○); Fim1p and Cdc8p together, spin (■,□); or Cdc8p, Fim1p, spin (◆,◇). (D) Plot of the fraction of Cdc8p in the low speed pellet (■), and the ratio of Cdc8p to actin in the low speed pellet (○), over a range of Fim1p concentrations.

Figure 2

Fim1p prevents Cdc8p from localizing to endocytic actin patches (A-B) In the absence of Fim1p, GFP-Cdc8p accumulates in ‘puncta’ at cell poles. (A) Distribution of GFP-Cdc8p in wild type (left) or fim1Δ (right) cells. Wedges and arrows indicate GFP-Cdc8p at the contractile ring and ‘puncta.’ Bar: 5 μm. (B) Quantification of GFP-Cdc8p ‘puncta’ fluorescence, determined from a line along the length of the cell (left), and plotted as a function of cell position (right; n=25 for each strain). Bar: 5 μm. (C-D) GFP-Cdc8p colocalizes with ARPC5-mCherry at actin patches in fim1Δ cells. (C) Z-projections of interphase wild type (left) and fim1Δ (right) cells. Bar: 5 μm. (D) Time-lapse micrographs of a single confocal section. Time is indicated (sec). (Top) ARPC5-mCherry. (Middle) GFP-Cdc8p. (Bottom) Merge. Wedges indicate actin patches. Bar: 1 μm. (See Movie S1) (E) GFP-Cdc8p is recruited to the contractile ring (wedge). (Left) Merge of interphase (arrow) and dividing (arrowhead) cells expressing ARPC5-mCherry and GFP-Cdc8p. Bar: 5 μm. (Right) Percent of ARPC5-mCherry patches containing GFP-Cdc8p in interphase and dividing cells (n=150 patches per condition).

Figure 3

Deletion of Fim1p causes endocytic actin patch defects (A-B) Endocytosis is impaired in fim1Δ cells. (A) DIC (left) and fluorescence (right) images of wild type (top) and fim1Δ (bottom) cells following 20 min incubation with 2 μM lipophilic dye FM4-64. Bar: 5 μm. (B) Plot of FM4-64 fluorescence uptake. Values are mean +/− standard deviation. (C-E) Actin patch dynamics are impaired in fim1Δ cells. (C) Time-lapse micrographs of single actin patches (wedges) in wild type (top) and fim1Δ (bottom) cells expressing the general actin marker GFP-CHD(rng2). Time (sec) is indicated at top. Bar: 0.5 μm. (See Movie S2) (D) Plot of actin patch lifetime for wild type and fim1Δ cells (n=40 patches for each strain). Values are mean +/− standard deviation. Asterisk: p<0.05 (E) Position over time (1.2 s intervals) for two representative wild type (left) and fim1Δ (right) actin patches. Dashed lines indicate cell cortex.

Figure 4

Actin patch dynamics in fim1Δ cells are recovered by depletion of Cdc8p (A-C) Wild type, cdc8-27, fim1Δ, and fim1Δ cdc8-27 cells expressing GFP-CHD(rng2) at 30°C. (A) Time-lapse micrographs (sec) of single actin patches (wedges) in the indicated cells. Bar: 1 μm. (See Movie S3) (B) Plot of average patch lifetime for the indicated cells (n=25 for each strain). Values are mean +/− standard deviation. Asterisk: p<0.05. (C) Position over time (0.25 s intervals) for two representative actin patches (black and orange). Dashed lines indicate cell cortex. (D) Plot of FM4-64 fluorescence uptake after 10 min at 30°C. Values are represented as mean +/− standard deviation.

Figure 5

Fim1p and Cdc8p have antagonistic roles during cytokinesis (A) Micrographs of the morphology (top) and nuclei and septa (bottom) of wild type, fim1Δ, cdc8-27 and fim1Δ cdc8-27 cells after 12 hours at 32°C. Bar: 5 μm. (B) Quantification of morphological phenotypes. Plots of cell area, abnormal septa (partial, misplaced, misaligned, and/or broad) and >2 nuclei (n =250 for each strain). Values are mean +/− standard deviation. (C-D) Effect of fim1Δ and cdc8-27 mutations on cytokinesis kinetics. Cells expressing GFP- CHD(rng2) at 30°C. (C) DIC and GFP time-lapse images of a representative wild type cell undergoing cytokinesis: (1) broad band detected, (2) contractile ring assembly complete, (3) ring constriction and septum ingression (arrow heads) begins, (4) constriction ends/septum complete, and (5) cells separate. Bar: 5 μm. (D) Plot of major cytokinesis events over time for at least 10 wild type (●), cdc8-27 (■), fim1Δ (◇) and fim1Δ cdc8-27 (△). Asterisk: p<0.05 for cdc8-27 compared to other strains.

Figure 6

Fim1p allows Adf1p-mediated severing in the presence Cdc8p (A-B) Effect of 100 μM Latrunculin A on F-actin in wild type, cdc8-27, fim1Δ, and fim1Δ cdc8-27 cells. (A) Micrographs before and 5 minutes after addition of Latrunculin A. Bar: 5 μm. (B) Average GFP-CHD(rng2) fluorescence at 20 cell poles. Values are mean +/− standard deviation. Asterisk: p<0.05. (C-D) TIRF microscopy observation of Adf1p-mediated severing. Bar: 5 μm. Initially, 1.5 μM Mg-ATP-actin (33% labeled with Oregon Green) was assembled in the presence of the indicated proteins. At time 0, 150 nM Adf1p was flowed into the chamber. (See Movie S4) (C) Time-lapse micrographs of the indicated reactions. Bar: 5 μm. (D) Plot of the severing rate (breaks μm⁻¹ sec⁻¹) in the presence of Fim1p and/or Cdc8p (n=15 filaments per condition). Values are mean +/− standard deviation. Asterisk: p<0.05. (E-F) Mutations in Fim1p and Adf1p have synergistic effects on actin patch dynamics. The indicated cells expressing GFP-CHD(rng2) at 25°C. (E) Time-lapse micrographs of representative actin patch (wedges) lifetimes (sec). Bar = 1.5 μm. (See Movie S5) (F) Plot of actin patch lifetime, binned in 20 second intervals (n=30 patches for each strain).

Figure 7

Cartoon model for regulation of Adf1p-mediated severing by Fim1p and Cdc8p in fission yeast.

1. At endocytic actin patches, high concentrations of Fim1p allow efficient Adf1p-mediated severing by preventing Cdc8p from binding to Arp2/3-nucleated actin filaments. In the absence of Fim1p, ectopic localization of Cdc8p to patches slows patch dynamics by inhibiting filament severing. Cdc8p may also inhibit actin filament nucleation by Arp2/3.

2. During contractile ring assembly, lower concentrations of Fim1p allow limited actin filament severing by partially inhibiting Cdc8p. Previous studies have suggested that severing is necessary for contractile ring assembly, despite the presence of high concentrations of Cdc8p [5, 21]