Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actinbinding protein, leiomodin, which acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed-end capping protein tropomodulin; a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity 3-fold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, suggesting a role for leiomodin in the nucleation of tropomyosindecorated filaments in muscles.Actin binding proteins suppress the spontaneous nucleation of actin monomers into filaments, so cells use nucleation factors to initiate actin polymerization. In non-muscle cells, the bestcharacterized filament nucleators are Arp2/3 complex and formins (1). Less is known about the initiation of actin filaments in striated and smooth muscle cells, where specialized proteins may be used to assemble and remodel the tropomyosin-decorated filaments.We identified leiomodin (Lmod) as a potential filament nucleator in muscle cells because sequence analysis suggested that it contained at least three actin-binding sites and could possibly recruit three actin monomers to form a polymerization nucleus. Thus, the first ~340 residues of Lmod are ~40% identical to tropomodulin (Tmod) ( fig. S1), a protein that caps actin filament pointed ends (2,3). The N-terminal portion of Tmod is unstructured, except for three helical segments involved in binding tropomyosin (residues 24-35 and 126-135) and actin (residues 65-75) (4). This region of Tmod, caps the pointed end of actin filaments in a tropomyosin-dependent manner (5). Tmod has a second, tropomyosin-independent, actinbinding and capping site within the C-terminal region (residues 160-359) (5), consisting almost †To whom correspondence should be addressed. droberto@mail.med.upenn.edu. 5 Present address: Elan Pharmaceuticals,

We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-Pi-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 M almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.filaments ͉ rate constant ͉ treadmilling A full understanding of actin dynamics in cells will require a complete set of kinetic and equilibrium constants for the polymerization and depolymerization reactions for the three different nucleotide states of actin monomers and polymers, which can bind ATP, ADP-P i (ADP with inorganic phosphate [P i ] bound noncovalently in the ␥-phosphate position), or ADP. Each of these species can bind and dissociate at both the fast growing barbed end and the slow growing pointed end of a filament. Monomeric actin hydrolyzes ATP very slowly (1), but polymerization changes the conformation of the subunits so that they hydrolyze ATP irreversibly (2) at 0.3 s Ϫ1 (3), assuming hydrolysis is random. The ␥-phosphate dissociates slowly from the ADP-P i intermediate with a half time of Ϸ350 s (4). The reaction is reversible, but the affinity of polymerized ADP-actin for inorganic phosphate is low, with a K d in the millimolar range (5). Electron microscopy (6, 7) and limited proteolysis (8) indicate that P i dissociation is coupled to a conformation change in the actin filament.As a result of ATP hydrolysis, single actin filaments at steady state add fresh ATP-subunits at the barbed end balanced by dissociation of ADP-actin at the pointed end in a process known as treadmilling (9-12). Treadmilling in a medium containing ATP depends on hydrolysis of ATP bound to polymerized subunits and P i dissociation coupled to differences in the rate constants for subunit association and dissociation at the two ends (9, 13). The critical concentrations of ATP-actin and ADP-P i -actin are similar (14, 15), but some of the kinetic parameters required to model the steady state behavior of actin (16) were unknown, such as the associati...

The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

SUMMARY Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets, but distinct from bundles formed by espin, an actin cross-linker in stereocilia. We generated mutant Triobp mice (TriobpΔex8/Δex8) that are profoundly deaf. Stereocilia of TriobpΔex8/Δex8 mice develop normally, but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.

Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CPcapped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weakcapping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there.cell migration | VASP

SUMMARY Latrunculin A (LatA), a toxin from the red sea sponge Latrunculia magnifica, is the most widely used reagent to depolymerize actin filaments in experiments on live cells. LatA binds actin monomers and sequesters them from polymerization [1, 2]. Low concentrations of LatA result in rapid (tens of seconds) disassembly of actin filaments in animal [3] and yeast cells [2]. Depolymerization is usually assumed to result from sequestration of actin monomers. Our observations of single muscle actin filaments by TIRF microscopy showed that LatA bound ATP-actin monomers with a higher affinity (Kd = 0.1 μM) than ADP-Pi-actin (Kd = 0.4 μM) or ADP-actin (Kd = 4.7 μM). LatA also slowly severed filaments and increased the depolymerization rate at both ends of filaments freshly assembled from ATP-actin to the rates of ADP-actin. This rate plateaued at LatA concentrations >60 μM. LatA did not change the depolymerization rates of ADP- actin filaments or ADP-Pi-actin filaments generated with 160 mM phosphate in the buffer. LatA did not increase the rate of phosphate release from bulk samples of filaments assembled from ATP-actin. Thermodynamic analysis showed that LatA binds weakly to actin filaments with a Kd >100 μM. We propose that concentrations of LatA much lower than this Kd promote phosphate dissociation only from both ends of filaments, resulting in depolymerization limited by the rate of ADP-actin dissociation. Thus, one must consider both rapid actin depolymerization and severing in addition to sequestering actin monomers when interpreting the effects of LatA on cells.

Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of ϳ90% at ϳ250 nM mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from ϳ30 min without mCAH3 to ϳ10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments. Capping protein (CP)2 is a highly conserved, ubiquitously expressed, heterodimeric actin-binding protein that plays a major role in limiting the duration of actin filament elongation within cells (1, 2). CP accomplishes this task by binding with high affinity (K d ϳ 0.1 nM) to the barbed end of the actin filament, thereby blocking the further association (and dissociation) of actin monomers at the fast growing end. CP is one of five proteins that are required for the reconstitution of actin polymerization-driven motility in vitro (3) Moreover, biochemical, cell biological, and modeling studies all suggest that rapid filament capping by CP in vivo is required in order to generate an Arp2/3-dependent dendritic actin network at the leading edge that is sufficiently branched to push the cell forward (4 -7). Consistent with these studies, cells that lack CP or in which CP levels have been reduced exhibit profound alterations in the assembly of their actin cytoskeleton (8 -12). Together, these observations highlight the necessity of identifying physiological regulators of CP function. A second compelling reason to search for such regulators stems from the very long half-time of CP at the barbed end in vitro (ϳ30 min for vertebrate CP) (13,14), because this half-time seems incompatible with the dynamics of actin in vivo, where large regions of F-actin ca...

Capping protein (CP) is a ubiquitously expressed, heterodimeric 62-kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. Myotrophin (V-1) is a 13-kDa ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here, we elucidate the molecular interaction between CP and V-1 by NMR. Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1/CP interaction, bind the basic patch near the joint of the ␣ tentacle of CP shown previously to drive most of the association of CP with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1. Capping protein (CP)3 is a ubiquitously expressed 62-kDa ␣/␤ heterodimer that binds the barbed end of the actin filament with high affinity (K d ϭ 0.1 nM) (1) to prevent further actin monomer association and dissociation, thereby limiting the extent of filament elongation in vivo (2, 3). In actin-based motility, such as that occurring in lamellipodia, new filaments are nucleated by the Arp2/3 complex to create a dendritic actin network at the leading edge. Biochemical, cell biological, and modeling studies all indicate that rapid filament capping by CP is required to sustain a dendritic network that is sufficiently branched to provide the motive force required for leading edge extension (4 -7). Consistent with its central role in actin network assembly, CP is one of only five proteins required for the reconstitution of actin-based motility in vitro (4,5,8), and cells lacking CP have profound deficiencies in actin cytoskeleton assembly (9 -13).Determination of the CP crystal structure led to the "tentacles" model of barbed end capping by CP (14). The two structurally homologous CP subunits form a central ␤-sheet that includes the bulk of the protein core, above which there are two antiparallel ␣-helices, one belonging to each subunit (14). At the end of these helices, each subunit contains a C-terminal "tentacle" which, on CP␣, is composed of an unstructured region punctuated in the middle by a short 4-residue helix, and on CP␤, it is composed of a longer amphipathic helix that protrudes from the protein core (Fig. 1). Based on crystallographic evidence, it was proposed that these C-terminal tentacles are flexible in solution, allowing them to bind and cap the barbed end. Extensive mutational studies in yeast (15) and vertebrate (1) CP that focused on the tentacles provided strong support for the tentacles model of capping. Specifically, deletion of the ␣ tentacle decreased the affinity of CP for the barbed end by 6,000-fold and its on-rate by 20-...