Actin Filament Nucleation Research Articles

Tropomyosin - master regulator of actin filament function in the cytoskeleton.

Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

Active Elastic Dimers: Cells Moving on Rigid Tracks

Experiments suggest that the migration of some cells in the three-dimensional extra cellular matrix bears strong resemblance to one-dimensional cell migration. Motivated by this observation, we construct and study a minimal one-dimensional model cell made of two beads and an active spring moving along a rigid track. The active spring models the stress fibers with their myosin-driven contractility and alpha-actinin-driven extendability, while the friction coefficients of the two beads describe the catch/slip bond behavior of the integrins in focal adhesions. In the absence of active noise, net motion arises from an interplay between active contractility (and passive extendability) of the stress fibers and an asymmetry between the front and back of the cell due to catch bond behavior of integrins at the front of the cell and slip bond behavior of integrins at the back. We obtain reasonable cell speeds with independently estimated parameters. We also study the effects of hysteresis in the active spring, due to catch bond behavior and the dynamics of cross-linking, and the addition of active noise on the motion of the cell. Our model highlights the role of alpha-actinin in three-dimensional cell motility and does not require Arp2/3 actin filament nucleation for net motion.

The Kinetics of Fesselin (Avian Synaptopodin 2) Binding to Smooth Muscle Myosin is Dependent on Calcium-Calmodulin

Fesselin (or avian synaptopodin 2) is an actin binding protein that nucleates actin filament formation (Leinweber et al. 1999; Beall et al. 2001) and bundles actin filaments (Schroeter et al. 2013). The nucleating activity is inhibited by Ca2+-calmodulin (Schroeter et al. 2004). Fesselin also binds myosin with an affinity of 2 x 106 M−1 at 50 mM ionic strength (Schroeter & Chalovich 2005). That binding increases the length and width of myosin filaments, and reduces the rate of dissociation of myosin filaments and actin-myosin complexes by ATP (Kingsbury et al. 2013). Fesselin labeled with IANBD produced a fluorescence increase upon binding to smooth muscle myosin or S1. The apparent rate constant for the fluorescence change increased with increasing concentrations of myosin or S1 in a hyperbolic fashion (105 mM ionic strength, 10 deg C). Binding to myosin began with the rapid formation of a low affinity intermediate followed by a second step having an observed rate constant (k2 + k-2) of 30/sec. Several observations suggested that the binding of fesselin to myosin was affected by calmodulin and calcium. The apparent rate constants for both processes in the binding of IANBD-fesselin to S1 were 2-fold faster in the absence of calcium. Binding of IANBD fesselin to intact myosin was complex in the presence of calcium with a rapid increase in fluorescence followed by a slower decrease; that behavior was not observed in the presence of calcium but absence of calmodulin. Ca2+-calmodulin may play roles in the formation of actin filaments, myosin filaments and actin-myosin complexes through fesselin.

Electrostatic Interactions between the Bni1p Formin FH2 Domain and Actin Influence Actin Filament Nucleation

Formins catalyze nucleation and growth of actin filaments. Here, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and showed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L, or both) reduced the interaction energies between the proteins, and in coarse-grained simulations, the formin lost more interprotein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins.

A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction.

Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.

Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.

Active elastic dimers: cells moving on rigid tracks.

Experiments suggest that the migration of some cells in the three-dimensional extracellular matrix bears strong resemblance to one-dimensional cell migration. Motivated by this observation, we construct and study a minimal one-dimensional model cell made of two beads and an active spring moving along a rigid track. The active spring models the stress fibers with their myosin-driven contractility and α-actinin-driven extendability, while the friction coefficients of the two beads describe the catch and slip-bond behaviors of the integrins in focal adhesions. In the absence of active noise, net motion arises from an interplay between active contractility (and passive extendability) of the stress fibers and an asymmetry between the front and back of the cell due to catch-bond behavior of integrins at the front of the cell and slip-bond behavior of integrins at the back. We obtain reasonable cell speeds with independently estimated parameters. We also study the effects of hysteresis in the active spring, due to catch-bond behavior and the dynamics of cross linking, and the addition of active noise on the motion of the cell. Our model highlights the role of α-actinin in three-dimensional cell motility and does not require Arp2/3 actin filament nucleation for net motion.

Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model.

BackgroundIntra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower.ResultsTo explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still to be verified in wet-lab experiments.ConclusionLetting cells grow on surface structures is a possibility to shed new light on the intricate mechanisms that relate membrane and actin related dynamics in the cell. Our results demonstrate the need for declarative expressive spatial modeling approaches that allow probing different hypotheses, and the central role of the focal adhesion complex not only for nucleating actin filaments, but also for regulating possible severing agents locally.

Structural analysis of the transitional state of Arp2/3 complex activation by two actin-bound WCAs

Actin filament nucleation and branching by Arp2/3 complex is activated by nucleation-promoting factors (NPFs), whose C-terminal WCA region contains binding sites for actin (W) and Arp2/3 complex (CA). It is debated whether one or two NPFs are required for activation. Here we present evidence in support of the two-NPF model and show that actin plays a crucial role in the interactions of two mammalian NPFs, N-WASP and WAVE2, with Arp2/3 complex. Competition between actin-WCA and glia maturation factor (GMF) for binding to Arp2/3 complex suggests that during activation the first actin monomer binds at the barbed end of Arp2. Based on distance constraints obtained by time-resolved fluorescence resonance energy transfer, we define the relative position of the two actin-WCAs on Arp2/3 complex and propose an atomic model of the 11-subunit transitional complex.

Formation of Transient Lamellipodia

Cell motility driven by actin polymerization is pivotal to the development and survival of organisms and individual cells. Motile cells plated on flat substrates form membrane protrusions called lamellipodia. The protrusions repeatedly appear and retract in all directions. If a lamellipodium is stabilized and lasts for some time, it can take over the lead and determine the direction of cell motion. Protrusions traveling along the cell perimeter have also been observed. Their initiation is in some situations the effect of the dynamics of the pathway linking plasma membrane receptors to actin filament nucleation, e.g. in chemotaxis. However, lamellipodia are also formed in many cells incessantly during motion with a constant state of the signaling pathways upstream from nucleation promoting factors (NPFs), or spontaneously in resting cells. These observations strongly suggest protrusion formation can also be a consequence of the dynamics downstream from NPFs, with signaling setting the dynamic regime but not initiating the formation of individual protrusions. A quantitative mechanism for this kind of lamellipodium dynamics has not been suggested yet. Here, we present a model exhibiting excitable actin network dynamics. Individual lamellipodia form due to random supercritical filament nucleation events amplified by autocatalytic branching. They last for about 30 seconds to many minutes and are terminated by filament bundling, severing and capping. We show the relevance of the model mechanism for experimentally observed protrusion dynamics by reproducing in very good approximation the repetitive protrusion formation measured by Burnette et al. with respect to the velocities of leading edge protrusion and retrograde flow, oscillation amplitudes, periods and shape, as well as the phase relation between protrusion and retrograde flow. Our modeling results agree with the mechanism of actin bundle formation during lamellipodium retraction suggested by Burnette et al. and Koestler et al.

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

3D Model of Cytokinetic Ring Assembly in Fission Yeast

During fission yeast cytokinesis, actin filaments are nucleated in random directions by cortical Cdc12 and captured by myosin-II motors from neighboring nodes, exerting forces that pull them together into a contractile ring. Contractile ring assembly has been previously described using a stochastic 2D computational model based on a “search, capture, pull and release” mechanism (SCPR). In this model, actin filaments are assumed to polymerize along the cell membrane, consistent with enhanced concentration of actin filaments near the cell membrane in experiments. Recent observations in mutant and wild-type cells have suggested that actin filaments nucleated in the cytoplasm and in non-medial locations can also participate into the assembly of the cytokinetic ring as well as in clump, star, and meshwork actomyosin morphologies. The relative contributions of the non-medially nucleated actin filaments has been debated. In order to examine these effects that involve 3D dynamics and to better understand the mechanisms that confine actin filaments along the cell surface, we extended the SCPR model to 3D. In this model semiflexible actin filaments (represented as beads connected by springs) grow from nodes and they can be captured by myosin in neighboring nodes. Cross-linking by alpha-actinin and fimbrin is represented by an attractive interaction between filament beads. We also implemented cytoplasmic formin nucleation and actin cable incorporation into the contractile ring. We quantified the resulting distribution and orientation of actin around the band of myosin nodes and studied the effects of varying model parameter values. We describe the conditions under which ring, clump, transient star and meshwork structures may form along the cell membrane and compare to prior experiments.

Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood

Successful completion of the Plasmodium lifecycle in the mosquito vector is critical for malaria transmission. It has been documented that the fate of Plasmodium in the mosquito ultimately depends on a fine interplay of molecular mosquito factors that act as parasite agonists and antagonists. Here we investigate whether the cellular responses of the invaded midgut epithelium can also determine the parasite fate and development. We show that the parasite hood, an actin-rich structure formed around the ookinete as it exits the epithelium, is a local epithelial defence reaction observed around 60% of invading parasites. The hood co-localizes with WASP, a promoter of actin filament nucleation, suggesting that it is an active reaction of the invaded cell against invading parasites. Importantly, depletion of WASP by RNAi leads to a significant reduction in hood formation, which is consistent with the previously documented role of this gene as a potent parasite antagonist. Indeed, in mosquitoes that are either genetically selected or manipulated by RNAi to be refractory to Plasmodium, most dead parasites exhibit an actin hood. In these mosquitoes, invading ookinetes are killed by lysis or melanization while exiting the midgut epithelium. Silencing WASP in these mosquitoes inhibits the formation of the hood and allows many parasites to develop to oocysts. These data in conjunction with fine microscopic observations suggest that the presence of the hood is linked to ookinete killing through lysis.

Enhanced fear expression in Spir-1 actin organizer mutant mice

Spir proteins nucleate actin filaments at vesicle membranes and facilitate intracellular transport processes. The mammalian genome encodes two Spir proteins, namely Spir-1 and Spir-2. While the mouse spir-2 gene has a rather broad expression pattern, high levels of spir-1 expression are restricted to the nervous system, oocytes, and testis. Spir-1 mutant mice generated by a gene trap method have been employed to address Spir-1 function during mouse development and in adult mouse tissues, with a specific emphasis on viability, reproduction, and the nervous system. The gene trap cassette disrupts Spir-1 expression between the N-terminal KIND domain and the WH2 domain cluster. Spir-1 mutant mice are viable and were born in a Mendelian ratio. In accordance with the redundant function of Spir-1 and Spir-2 in oocyte maturation, spir-1 mutant mice are fertile. The overall brain anatomy of spir-1 mutant mice is not altered and visual and motor functions of the mice remain normal. Microscopic analysis shows a slight reduction in the number of dendritic spines on cortical neurons. Detailed behavioral studies of the spir-1 mutant mice, however, unveiled a very specific and highly significant phenotype in terms of fear learning in male mice. In contextual and cued fear conditioning experiments the male spir-1 mutant mice display increased fear memory when compared to their control littermates. Our data point toward a particular function of the vesicle associated Spir-1 actin organizer in neuronal circuits determining fear behavior.

Direct interaction between two actin nucleators is required in Drosophila oogenesis

Controlled actin assembly is crucial to a wide variety of cellular processes, including polarity establishment during early development. The recently discovered actin mesh, a structure that traverses the Drosophila oocyte during mid-oogenesis, is essential for proper establishment of the major body axes. Genetic experiments indicate that at least two proteins, Spire (Spir) and Cappuccino (Capu), are required to build this mesh. The spire and cappuccino genetic loci were first identified as maternal effect genes in Drosophila. Mutation in either locus results in the same phenotypes, including absence of the mesh, linking them functionally. Both proteins nucleate actin filaments. Spir and Capu also interact directly with each other in vitro, suggesting a novel synergistic mode of regulating actin. In order to understand how and why proteins with similar biochemical activity would be required in the same biological pathway, genetic experiments were designed to test whether a direct interaction between Spir and Capu is required during oogenesis. Indeed, data in this study indicate that Spir and Capu must interact directly with one another and then separate to function properly. Furthermore, these actin regulators are controlled by a combination of mechanisms, including interaction with one another, functional inhibition and regulation of their protein levels. Finally, this work demonstrates for the first time in a multicellular organism that the ability of a formin to assemble actin filaments is required for a specific structure.

IQGAP1 interactome analysis by in vitro reconstitution and live cell 3‐color FRET microscopy

IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Förster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1, and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells.

Direct interaction between two actin nucleators is required in Drosophila oogenesis

Controlled actin assembly is crucial to a wide variety of cellular processes, including polarity establishment during early development. The recently discovered actin mesh, a structure that traverses the Drosophila oocyte during mid-oogenesis, is essential for proper establishment of the major body axes. Genetic experiments indicate that at least two proteins, Spire (Spir) and Cappuccino (Capu), are required to build this mesh. The spire and cappuccino genetic loci were first identified as maternal effect genes in Drosophila. Mutation in either locus results in the same phenotypes, including absence of the mesh, linking them functionally. Both proteins nucleate actin filaments. Spir and Capu also interact directly with each other in vitro, suggesting a novel synergistic mode of regulating actin. In order to understand how and why proteins with similar biochemical activity would be required in the same biological pathway, genetic experiments were designed to test whether a direct interaction between Spir and Capu is required during oogenesis. Indeed, data in this study indicate that Spir and Capu must interact directly with one another and then separate to function properly. Furthermore, these actin regulators are controlled by a combination of mechanisms, including interaction with one another, functional inhibition and regulation of their protein levels. Finally, this work demonstrates for the first time in a multicellular organism that the ability of a formin to assemble actin filaments is required for a specific structure.

The Bacterial Effector VopL Organizes Actin into Filament-like Structures

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

The formin protein mDia2 serves as a marker of spindle pole dynamics in vitrified-warmed mouse oocytes.

The mouse diaphanous 2 (mDia2) protein belongs to the formin family and has been shown to nucleate actin filaments and stabilize microtubules, thus indicating a role in cytoskeleton organization. Our previous study, which showed that mDia2 specifically localizes to spindle poles of metaphase I mouse oocytes and NIH3T3 cells, provided the first evidence of its spindle pole-associated cellular function. In the present study, we aim to determine whether spindle pole proteins, such as mDia2 and pericentrin, can be used to monitor the status of spindle poles in cryopreserved mouse oocytes. We show herein that mDia2 exhibits an overlapping distribution with pericentrin, which is a crucial component of centrosomes and microtubule organizing centers (MTOCs). In vitrified-warmed oocytes, the overlapping distribution of mDia2 and pericentrin was immediately detected after thawing, thereby suggesting that mDia2 maintains a tight association with the spindle pole machinery. Interestingly, we observed that microtubules extend from mDia2 clusters in cytoplasmic MTOCs after thawing. This result suggests that mDia2 is a major MTOC component that is closely associated with pericentrin and that it plays a role in microtubule growth from MTOCs. Collectively, our results provide evidence that mDia2 is a novel marker of spindle pole dynamics before and after cryopreservation.

Arp2/3 complex inhibitors adversely affect actin cytoskeleton remodeling in the cultured murine kidney collecting duct M-1 cells

Dynamic remodeling of the actin cytoskeleton plays an essential role in cell migration and various signaling processes in living cells. One of the critical factors that controls the nucleation of new actin filaments in eukaryotic cells is the actin-related protein 2/3 (Arp2/3) complex. Recently, two novel classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate F-actin have been discovered and described. The current study aims at investigating the effects of CK-0944666 (CK-666) and its analogs (CK-869 and inactive CK-689) on the reorganization of the actin microfilaments in the cortical collecting duct cell line, M-1. We show that treatment with CK-666 and CK869 results in the reorganization of F-actin and drastically affects cell motility rate. The concentrations of the compounds used in this study (100-200 μM) neither cause loss of cell viability nor influence cell shape or monolayer integrity; hence, the effects of described compounds were not due to structural side effects. Therefore, we conclude that the Arp2/3 complex plays an important role in cell motility and F-actin reorganization in M-1 cells. Furthermore, CK-666 and its analogs are useful tools for the investigation of the Arp2/3 complex.