James B. Moseley

The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism

SUMMARY All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action.

Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6.

Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop.

Regulated Binding of Adenomatous Polyposis Coli Protein to Actin

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. Here we show that purified carboxyl-terminal basic domain of human APC protein (APC-basic) bound directly to and bundled actin filaments and associated with actin stress fibers in microinjected cells. Actin filaments and microtubules competed for binding to APC-basic, but APC-basic also could cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro were inhibited by its ligand EB1, and co-microinjection of EB1 prevented APC association with stress fibers. Point mutations in EB1 that disrupted APC binding relieved the inhibition in vitro and restored APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression.

Twinfilin is an actin-filament-severing protein and promotes rapid turnover of actin structures in vivo

Working in concert, multiple actin-binding proteins regulate the dynamic turnover of actin networks. Here, we define a novel function for the conserved actin-binding protein twinfilin, which until now was thought to function primarily as a monomer-sequestering protein. We show that purified budding yeast twinfilin (Twf1) binds to and severs actin filaments in vitro at pH below 6.0 in bulk kinetic and fluorescence microscopy assays. Further, we use total internal reflection fluorescence (TIRF) microscopy to demonstrate that Twf1 severs individual actin filaments in real time. It has been shown that capping protein directly binds to Twf1 and is required for Twf1 localization to cortical actin patches in vivo. We demonstrate that capping protein directly inhibits the severing activity of Twf1, the first biochemical function ascribed to this interaction. In addition, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] inhibits Twf1 filament-severing activity. Consistent with these biochemical activities, a twf1Δ mutation causes reduced rates of cortical actin patch turnover in living cells. Together, our data suggest that twinfilin coordinates filament severing and monomer sequestering at sites of rapid actin turnover and is controlled by multiple regulatory inputs.

Displacement of Formins from Growing Barbed Ends by Bud14 Is Critical for Actin Cable Architecture and Function

Normal cellular development and function require tight spatiotemporal control of actin assembly. Formins are potent actin assembly factors that protect the growing ends of actin filaments from capping proteins. However, it is unresolved how the duration of formin-mediated actin assembly events is controlled, whether formins are actively displaced from growing ends, and how filament length is regulated in vivo. Here, we identify Bud14 as a high-affinity inhibitor of the yeast formin Bnr1 that rapidly displaces the Bnr1 FH2 domain from growing barbed ends. Consistent with these activities, bud14Delta cells display fewer actin cables, which are aberrantly long, bent, and latrunculinA resistant, leading to defects in secretory vesicle movement. Moreover, bud14Delta suppressed mutations that cause abnormally numerous and shortened cables, restoring wild-type actin architecture. From these results, we propose that formin displacement factors regulate filament length and are required in vivo to maintain proper actin network architecture and function.

Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor

Bud6 functions as an actin nucleation–promoting factor (NPF) for Bni1; thus formins can depend on NPFs like the Arp2/3 complex. Unexpected parallels exist between Bud6 and WASp. Bud6 is the first nonmetazoan example of formins pairing with actin monomer–binding proteins to stimulate nucleation, akin to Spire-Capu and APC-mDia1