Three's company: the fission yeast actin cytoskeleton

Abstract

How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly challenging in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.

Figure 1. Overview of the three major actin structures in fission yeast

(a) Fluorescent image of the actin cytoskeleton in a population of fission yeast cells expressing the general F-actin marker GFP-CHD (calponin homology domain of Rng2). (b) Cartoon summarizing the subcellular distribution of actin structures during the cell cycle (centering on mitosis). The table below highlights the basic features and roles of the three actin structures. (c) Venn diagram summarizing the localization of highly conserved actin-binding proteins across actin patches (blue), actin cables (green) and the contractile ring (red). Actin-binding proteins are listed under generic and fission yeast protein names in groups outside the diagram based on their cellular distribution. Within the diagram, proteins overlapping two or more structures (black text) are further categorized using colored + signs to emphasize relative protein levels in each actin structure. Arp2/3 complex: consists of seven different subunits. Capping protein: heterodimer of Acp1 and Acp2. Hip1R: Huntingtin interacting protein-related, talin-like. Myosins and IQGAP associate with light chains (Myo1, calmodulin and Cam2; Myo2 and Myp2, Cdc4 and Rlc1; Myo51, calmodulin and Cdc4; Myo52, calmodulin; Rng2, calmodulin and Cdc4). (d) Regulation of actin filament turnover and myosin motors by tropomyosin and fimbrin , . Actin patches: High concentrations of fimbrin Fim1 prevent tropomyosin Cdc8 from binding the Arp2/3 complex-nucleated branched filaments, which allows efficient cofilin Cof1-mediated actin filament turnover and recruitment of myosin-I Myo1. Actin cables: tropomyosin favors myosin-V Myo52-directed motility on formin For3-nucleated straight parallel filaments. Contractile rings: Lower concentrations of fimbrin allow limited cofilin severing by partially inhibiting tropomyosin. Tropomyosin also favors myosin-II Myo2-mediated compaction of the formin Cdc12-nucleated straight antiparallel filaments.

Figure 2. Endocytic actin patches

(a) Time course of endocytic actin patch assembly. Clathrin arrives at patches 100 seconds before internalization and endocytic vesicle scission at time zero, and is accompanied by early endocytic proteins(Early). Endocytic adaptor proteins (Adaptors) are recruited into patches 30–40 s before internalization. The Arp2/3 complex activators, Wsp1-Vrp1 complex and myosin-I Myo1, appear in patches 10 seconds before internalization and are accompanied by proteins thought to regulate their activity (Regulators). Arp2/3 complex is recruited a few seconds after Wsp1 and stimulates a burst of actin assembly and recruitment of actin-binding proteins. Coronin Crn1 arrives with a 5 second delay. F-BAR proteins (BAR) may cooperate with actin in promoting membrane invagination. Upon patch internalization, Myo1 stays behind, clathrin and Wsp1-Vrp1 rapidly dissipate, and some patches associate with actin cables and undergo retrograde flow. (b) Dendritic nucleation model of actin patch assembly and disassembly. Branched filament arrangement is inferred from the in vitro properties of the Arp2/3 complex, and the ultrastructure of budding yeast patches , , . (1) Inactive Wsp1 is recruited to the patch and activated; (2) Wsp1 binds actin monomer; (3) Wsp1-actin binds Arp2/3 complex; (4) ternary complex of Wsp1-actin-Arp2/3 complex binds to the side of actin filament; (5) Arp2/3 complex is activated and nucleates actin filament branch; (6) branch elongates; (7) capping protein Acp1/2 caps filaments; (8) fimbrin Fim1 cross-links filaments; (9) coronin Crn1 binds to filaments; (10) cofilin Cof1 severs filaments; (11) filament fragments diffuse away; (12) Cof1, Aip1, Srv2/CAP and profilin Cdc3 cooperate to disassemble filaments and recycle actin monomers. (c) Patches and contractile actin rings are distinct structures. The image is a 3D reconstruction of Z-series of spinning disk confocal images of red actin patch marker Cam2-mCherry (Myo1 light chain) and green actin ring marker Rlc1-mGFP (Myo2 light chain) in, from top to bottom, a cell with a broad band of pre-ring nodes, a cell with an unconstricted ring, and a cell with a constricting ring. Scale bar, 1 µm.

Figure 3. Mechanisms of contractile ring assembly

(a) Schematic of a fission yeast cell illustrating the timing of contractile ring (red) assembly, dwell/maturation, and constriction relative to spindle elongation (black) and SIN activity during mitosis. (b) An adaptation of the ‘search, capture, pull, and release’ mechanism of ring assembly . Mid1 providesthe spatial cue for Rng2 and Myo2. Rng3 ensures that Myo2 motors are active. Cdc15 and Cdc12 arrive and stimulate actin assembly. Cdc8 binds filaments and promotes Cdc12-mediated actin elongation, limits cofilin Cof1-mediated filament severing, and specifies Myo2 motor activity leading to actomyosin ring compaction. (c) Schematic summarizing ring assembly from actomyosin cables (red) by the SIN-dependent ‘leading cable’ mechanism. The contribution of this mechanism to ring assembly becomes most apparent in a mid1Δ cps1-191 double mutant when the spatial organization of Mid1-dependent nodes is removed and septum formation is delayed by the cps1 mutant . This delay ensures that ring assembly finishes in time to provide a tight spatial landmark for deposition of the septum. A mechanism by which the SIN communicates with the ring is outlined (inset): SIN promotes Clp1 phosphatase activity in the cytoplasm which (along with other unidentified phosphatases) dephosphorylates Cdc15 –. Dephosphorylation promotes self-assembly, membrane deformation, recruitment of formin Cdc12 , and associations with paxillin Pxl1 and C2 domain protein Fic1 to promote ring stability .

Figure 4. Actin cable assembly in fission yeast

(a) Fluorescent image of fission yeast expressing the general F-actin marker GFP-CHD, and a corresponding cartoon diagram of microtubules and actin cables in a single cell. Interphase cells contain ~four dynamic microtubules whose plus ends periodically interact with the cell tip, and deliver polarity factors that direct actin cable assembly . Actin cables are polarized tracks utilized by myosin-V motors to deliver materials to cell tips , . Actin cables are bundles of short parallel actin filaments assembled by the formin For3 –. (b) Model for actin cable assembly. A-Inactive For3 diffuses to the cell tip (For3: 1 and 2). B-Microtubules deposit the +TIP Tea1/Tea4 complex at cell ends where it recruits the polarisome complex, which includes For3 and its activators Rho-GTPase Cdc42, Bud6 and Pob1 , , . C-Activated For3 (1) transiently mediates processive actin filament assembly. D-Seconds later For3 (1) is partially inactivated, remains filament-bound but does not facilitate further elongation, and releases from the cortex along with its associated short filament. D and E-Activation and transient processive actin filament assembly by a neighboring For3 (2) pushes partially active For3 molecules and associated filaments inward by retrograde flow as the cable grows . F-Actin cables are disassembled distal to the cell tip by an unknown mechanism, and inactive For3 and actin monomers are subsequently recycled back to the tip.

Figure I. Fission yeast mating

Figure II. In vivo methodology

Figure III. In vitro methodology