Endocytic Actin Patches Research Articles

Mechanism of a Concentration-dependent Switch between Activation and Inhibition of Arp2/3 Complex by Coronin

Arp2/3 complex is a key actin filament nucleator that assembles branched actin networks in response to cellular signals. The activity of Arp2/3 complex is regulated by both activating and inhibitory proteins. Coronins make up a large class of actin-binding proteins previously shown to inhibit Arp2/3 complex. Although coronins are known to play a role in controlling actin dynamics in diverse processes, including endocytosis and cell motility, the precise mechanism by which they regulate Arp2/3 complex is unclear. We conducted a detailed biochemical analysis of budding yeast coronin, Crn1, and found that it not only inhibits Arp2/3 complex but also activates it. We mapped regions required for activation and found that Crn1 contains a sequence called CA, which is conserved in WASp/Scar proteins, the prototypical activators of Arp2/3 complex. Point mutations in CA abolished activation of Arp2/3 complex by Crn1 in vitro. Confocal microscopy and quantitative actin patch tracking showed that these mutants had defective endocytic actin patch dynamics in Saccharomyces cerevisiae, indicating that activation of Arp2/3 complex by coronin is required for normal actin dynamics in vivo. The switch between the dual modes of regulation by Crn1 is controlled by concentration, and low concentrations of Crn1 enhance filament binding by Arp2/3 complex, whereas high concentrations block binding. Our data support a direct tethering recruitment model for activation of Arp2/3 complex by Crn1 and suggest that Crn1 indirectly inhibits Arp2/3 complex by blocking it from binding actin filaments.

Purification of Actin from Fission Yeast Schizosaccharomyces pombe and Characterization of Functional Differences from Muscle Actin

Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.

Reconstitution and Protein Composition Analysis of Endocytic Actin Patches

Clathrin-actin-mediated endocytosis in yeast involves the progressive assembly of at least 60 different proteins at cortical sites. More than half of these proteins are involved in the assembly of a branched network of actin filaments to provide the forces required for plasma membrane invagination. To gain insights into the regulation of endocytic actin patch dynamics, we developed an in vitro actin assembly assay using microbeads functionalized with the nucleation promoting factor (NPF) Las17 (yeast WASP). When incubated in a yeast extract, these beads assembled actin networks, and a significant fraction became motile. Multidimensional protein identification technology (MudPIT) showed that the recruitment of actin-binding proteins to these Las17-derived actin networks is selective. None of the proteins known to exclusively regulate the in vivo formation of actin cables or the actin contractile ring were identified. Our analysis also identified components of three other cortical structures, eisosomes, phosphoinositide kinase (PIK) patches, and the TORC2 complex, establishing intriguing biochemical connections between four different yeast cortical complexes. Finally, we identified Aim3 as a regulator of actin dynamics at endocytic sites. WASP is sufficient to trigger assembly of actin networks composed selectively of actin patch proteins. These experiments establish that the protein composition of different F-actin structures is determined by the protein factor that initiates the network. The identification of binding partners revealed new biochemical connections between WASP-derived networks and other cortical complexes and identified Aim3 as a novel regulator of the endocytic actin patch.

Differential Regulation of Unconventional Fission Yeast Myosins via the Actin Track

Fission yeast possesses three unconventional myosins: Myo1p (a class I myosin that functions at endocytic actin patches) and Myo51p and Myo52p (class V myosins that function at contractile rings and actin cables, respectively). Here we used a combination of in vivo and in vitro approaches to investigate how changes in the actin track influence the motor activity and spatial regulation of these myosins. We optimized the isolation of Myo1p, Myo51p, and Myo52p. All three myosins exhibited robust motor activity in ATPase and actin filament gliding assays. However, decoration of actin with tropomyosin differentially regulates the activity of these motors. Tropomyosin inhibits Myo1p by blocking its ability to form productive associations with actin filaments, whereas tropomyosin increases the actin affinity and ATPase activity of Myo51p and Myo52p. The actin filament crosslinking protein fimbrin rescues Myo1p motor activity by displacing tropomyosin from actin filaments. Consistent with our in vitro findings, fimbrin and tropomyosin have opposing effects on Myo1p function at actin patches. Defects in tropomyosin function led to shorter Myo1p patch lifetimes, whereas loss of fimbrin extended Myo1p lifetimes. Furthermore, defects in tropomyosin function decreased the efficiency of Myo52p-directed motility along actin cables in the cell. Tropomyosin promotes myosin-V motility along actin cables. Accumulation of fimbrin at actin patches relieves Myo1p from tropomyosin-mediated inhibition, ensuring maximal myosin-I motor activity at these sites. Thus, spatial regulation of myosin motor function is in part controlled by specific changes in the composition of the actin track.

Fimbrin and Tropomyosin Competition Regulates Endocytosis and Cytokinesis Kinetics in Fission Yeast

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. 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. 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.

Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and gamma-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010, MBoC 21: 2792-2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.

Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

The BAR Domain Superfamily: Membrane-Molding Macromolecules

Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.

Distinct Roles for Arp2/3 Regulators in Actin Assembly and Endocytosis

The Arp2/3 complex is essential for actin assembly and motility in many cell processes, and a large number of proteins have been found to bind and regulate it in vitro. A critical challenge is to understand the actions of these proteins in cells, especially in settings where multiple regulators are present. In a systematic study of the sequential multicomponent actin assembly processes that accompany endocytosis in yeast, we examined and compared the roles of WASp, two type-I myosins, and two other Arp2/3 activators, along with that of coronin, which is a proposed inhibitor of Arp2/3. Quantitative analysis of high-speed fluorescence imaging revealed individual functions for the regulators, manifested in part by novel phenotypes. We conclude that Arp2/3 regulators have distinct and overlapping roles in the processes of actin assembly that drive endocytosis in yeast. The formation of the endocytic actin patch, the creation of the endocytic vesicle, and the movement of the vesicle into the cytoplasm display distinct dependencies on different Arp2/3 regulators. Knowledge of these roles provides insight into the in vivo relevance of the dendritic nucleation model for actin assembly.