Henry N. Higgs

The many faces of actin: matching assembly factors with cellular structures

Actin filaments are major components of at least 15 distinct structures in metazoan cells. These filaments assemble from a common pool of actin monomers, but do so at different times and places, and in response to different stimuli. All of these structures require actin-filament assembly factors. To date, many assembly factors have been identified, including Arp2/3 complex, multiple formin isoforms and spire. Now, a major task is to figure out which factors assemble which actin-based structures. Here, we focus on structures at the plasma membrane, including both sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli). Insights gained from studies of adherens junctions and the immunological synapse are also considered.

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott–Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

Control of the Assembly of ATP- and ADP-Actin by Formins and Profilin

Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.

The Mouse Formin mDia1 Is a Potent Actin Nucleation Factor Regulated by Autoinhibition

Formin proteins are widely expressed in eukaryotes and play essential roles in assembling specific cellular actin-based structures. Formins are defined by a Formin Homology 2 (FH2) domain, as well as a proline-rich FH1 domain that binds the actin monomer binding protein, profilin, and other ligands. Constructs including FH2 of budding yeast Bni1 or fission yeast Cdc12 formins nucleate actin filaments in vitro. In this study, we demonstrate that FH2-containing constructs of murine mDia1 (also called p140 mDia or Drf1) are much more potent actin nucleators than the yeast formins. FH1 is necessary for nucleation when actin monomers are profilin bound. mDia1 is a member of the Diaphanous formin subfamily (Dia), whose members contain an N-terminal Rho GTPase binding domain (GBD) and a C-terminal Diaphanous autoinhibitory domain (DAD, ). Based on cellular and in vitro binding studies, an autoinhibitory model for Dia formin regulation proposes that GBD binding to DAD inhibits Dia-induced actin remodeling, whereas Rho binding activates by releasing GBD from DAD. Supporting this model, our results show that an N-terminal mDia1 construct strongly inhibits actin nucleation by the C terminus. RhoA partially relieves inhibition but does so when bound to either GDP or GTP analogs. Both N- and C-terminal mDia1 constructs appear to be multimeric.

An Actin-Dependent Step in Mitochondrial Fission Mediated by the ER-Associated Formin INF2

Masterminding Mitochondrial Fission Mitochondria are highly dynamic and undergo fusion and fission and they move in cells. Defects in mitochondrial dynamics are implicated in many neurodegenerative diseases. Recent findings have suggested that mitochondrial fission occurs preferentially at endoplasmic reticulum (ER) contact sites, with ER circumscribing mitochondria and possibly promoting the constriction of mitochondria during fission. Korobova et al. (p. 464) now suggest that an ER-localized formin, INF2, is required for mitochondrial fission and that INF2-mediated actin polymerization facilitates mitochondrial constriction. Actin filaments between the endoplasmic reticulum and mitochondria promote mitochondrial fission. Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.

Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a pattern of kidney injury observed either as an idiopathic finding or as a consequence of underlying systemic conditions. Several genes have been identified that, when mutated, lead to inherited FSGS and/or the related nephrotic syndrome. These findings have accelerated the understanding of glomerular podocyte function and disease, motivating our search for additional FSGS genes. Using linkage analysis, we identified a locus for autosomal-dominant FSGS susceptibility on a region of chromosome 14q. By sequencing multiple genes in this region, we detected nine independent nonconservative missense mutations in INF2, which encodes a member of the formin family of actin-regulating proteins. These mutations, all within the diaphanous inhibitory domain of INF2, segregate with FSGS in 11 unrelated families and alter highly conserved amino acid residues. The observation that alterations in this podocyte-expressed formin cause FSGS emphasizes the importance of fine regulation of actin polymerization in podocyte function.

Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex

The Wiskott–Aldrich-syndrome protein (WASP) regulates polymerization of actin by the Arp2/3 complex. Here we show, using fluorescence anisotropy assays, that the carboxy-terminal WA domain of WASP binds to a single actin monomer with a Kd of 0.6 μM in an equilibrium with rapid exchange rates. Both WH-2 and CA sequences contribute to actin binding. A favourable ΔH of −10 kcal mol−1 drives binding. The WA domain binds to the Arp2/3 complex with a Kd of 0.9 μM; both the C and A sequences contribute to binding to the Arp2/3 complex. Wiskott–Aldrich-syndrome mutations in the WA domain that alter nucleation by the Arp2/3 complex over a tenfold range without affecting affinity for actin or the Arp2/3 complex indicate that there may be an activation step in the nucleation pathway. Actin filaments stimulate nucleation by producing a fivefold increase in the affinity of WASP-WA for the Arp2/3 complex.

Regulation of Actin Polymerization by Arp2/3 Complex and WASp/Scar Proteins

Actin polymerization is required for many types of cell motility, such as chemotaxis, nerve growth cone movement, cell spreading, and platelet activation (reviewed in Ref. 1). In the lamellipodia that push forward the leading edge of motile cells, polymerizing filaments form a meshwork consisting of “Y branches” with the pointed end of one filament attached to the side of another filament (2). This meshwork presumably may provide a rigid body against which polymerization can drive membrane protrusion (3). A major unanswered question is how cells integrate signals coming through a variety of pathways to control when and where actin polymerizes. The filaments grow from a huge pool of unpolymerized actin maintained by monomer-binding proteins at a concentration approximately 1000-fold higher than required for spontaneous polymerization of actin (reviewed in Ref. 4). The monomerbinding protein profilin biases the direction of filament elongation, allowing growth at the fast growing barbed end but not the slow growing pointed end (reviewed in Ref. 4). In cells capping proteins block the barbed end of most filaments, so some mechanism is required to start new filaments (5). Cells might trigger actin polymerization in three ways: 1) de novo nucleation of filaments from monomeric actin; 2) severing existing filaments to create uncapped barbed ends; and 3) uncapping existing barbed ends. There is evidence for each of these mechanisms in various cellular processes, but new filaments are often created during cell motility (6), placing emphasis on mechanisms 1 and 2. Although activation of de novo nucleation by cell stimulation has long been an attractive model (7), no barbed end nucleating factors were known until it was discovered that Arp2/3 complex promotes actin nucleation, creating filaments that grow at their barbed ends (8). Because nucleation is rate-limiting in actin polymerization and strongly suppressed by monomer-binding proteins, Arp2/3 complex may be a key mediator of actin polymerization in cells. Arp2/3 complex also cross-links actin filaments end-to-side, indistinguishable from the Y branches at the leading edge (8). Based on these biochemical activities, Mullins et al. (8) proposed the dendritic nucleation model, whereby Arp2/3 complex both creates new filaments and cross-links them into a branching meshwork. Cellular observations support this model. Arp2/3 complex is concentrated at the leading edge of motile cells (9–13), specifically at the junctions of the Y branches (12, 13). It exists in all eukaryotes examined, and ablation of Arp2/3 complex subunits in Saccharomyces cerevisiae and Schizosaccharomyces pombe is lethal or severely debilitating (14–21). The next breakthrough was the discovery that ActA, a cell surface protein from the pathogenic bacterium, Listeria monocytogenes, stimulates Arp2/3 complex to nucleate actin in vitro (22). Listeria uses force generated by actin polymerization to propel itself around the cytoplasm of eukaryotic cells. ActA is the only bacterial protein required to induce polymerization, but ActA cannot stimulate actin filament formation by itself (reviewed in Ref. 23). This work suggested that cellular factors might activate Arp2/3 complex to nucleate actin. This year WASp/Scar proteins were identified as the first example of such factors (24–28). These proteins also interact with a variety of cell signaling molecules known to influence cytoskeletal dynamics, bringing us closer to forging a connection between surface receptor stimulation and actin polymerization. The rapid progress reviewed here depended upon groundwork from many laboratories. Analysis of Wiskott-Aldrich syndrome protein (WASp) and its neural homolog N-WASP revealed a binding site for Rho family GTPases and other domains that affect actin assembly in cells (29, 30). Study of GTPgS-stimulated actin polymerization in extracts of vertebrate cells (31–34), Dictyostelium (33), and Acanthamoeba (35) demonstrated that the Rho family GTPase Cdc42 mediates the effect of GTP and that Arp2/3 complex is required. Similar experiments with extracted yeast suggested that Bee1p (a WASp homolog) and Arp2/3 complex are required for actin patch assembly (20, 36, 37).

Dissecting Requirements for Auto-inhibition of Actin Nucleation by the Formin, mDia1

The mammalian formin, mDia1, is an actin nucleation factor. Experiments in cells and in vitro show that the N-terminal region potently inhibits nucleation by the formin homology 2 (FH2) domain-containing C terminus and that RhoA binding to the N terminus partially relieves this inhibition. Cellular experiments suggest that potent inhibition depends upon the presence of the diaphanous auto-regulatory domain (DAD) C-terminal to FH2. In this study, we examine in detail the N-terminal and C-terminal regions required for this inhibition and for RhoA relief. Limited proteolysis of an N-terminal construct from residues 1–548 identifies two stable truncations: 129–548 and 129–369. Analytical ultracentrifugation suggests that 1–548 and 129–548 are dimers, whereas 129–369 is monomeric. All three N-terminal constructs inhibit nucleation by the full C terminus. Although inhibition by 1–548 is partially relieved by RhoA, inhibition by 129–548 or 129–369 is RhoA-resistant. At the C terminus, DAD deletion does not affect nucleation but decreases inhibitory potency of 1–548 by 20,000-fold. Synthetic DAD peptide binds both 1–548 and 129–548 with similar affinity and partially relieves nucleation inhibition. C-terminal constructs are stable dimers. Our conclusions are as follows: 1) DAD is an affinity-enhancing motif for auto-inhibition; 2) an N-terminal domain spanning residues 129–369 (called DID for diaphanous inhibitory domain) is sufficient for auto-inhibition; 3) a dimerization region C-terminal to DID increases the inhibitory ability of DID; and 4) DID alone is not sufficient for RhoA relief of auto-inhibition, suggesting that sequences N-terminal to DID are important to RhoA binding. An additional finding is that FH2 domain-containing constructs of mDia1 and mDia2 lose >75% nucleation activity upon freeze-thaw.

INF2 Is a WASP Homology 2 Motif-containing Formin That Severs Actin Filaments and Accelerates Both Polymerization and Depolymerization

Formin proteins modulate both nucleation and elongation of actin filaments through processive movement of their dimeric formin homology 2 (FH2) domains with filament barbed ends. Mammals possess at least 15 formin genes. A subset of formins termed “diaphanous formins” are regulated by autoinhibition through interaction between an N-terminal diaphanous inhibitory domain (DID) and a C-terminal diaphanous autoregulatory domain (DAD). Here, we found several striking features for the mouse formin, INF2. First, INF2 interacted directly with actin through a region C-terminal to the FH2. This second interacting region sequesters actin monomers, an activity that is dependent on a WASP homology 2 (WH2) motif. Second, the combination of the FH2 and C-terminal regions of INF2 resulted in its curious ability to accelerate both polymerization and depolymerization of actin filaments. The mechanism of the depolymerization activity, which is novel for formin proteins, involves both the monomer binding ability of the WH2 and a potent severing activity that is dependent on covalent attachment of the FH2 to the C terminus. Phosphate inhibits both the depolymerization and severing activities of INF2, suggesting that phosphate release from actin subunits in the filament is a trigger for depolymerization. Third, INF2 contains an N-terminal DID, and the WH2 motif likely doubles as a DAD in an autoinhibitory interaction.