Filamin protects myofibrils from contractile damage through changes in its mechanosensory region.

Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion.

Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

TRANSPARENT TESTA GLABRA2 defines trichome cell shape by modulating actin cytoskeleton in Arabidopsis thaliana.

The Arabidopsis (Arabidopsis thaliana) TRANSPARENT TESTA GLABRA2 (TTG2) gene encodes a WRKY transcription factor that regulates a range of development events like trichome, seed coat, and atrichoblast formation. Loss-of-function of TTG2 was previously shown to reduce or eliminate trichome specification and branching. Here, we report the identification of an allele of TTG2, ttg2-6. In contrast to the ttg2 mutants described before, ttg2-6 displayed unique trichome phenotypes. Some ttg2-6 mutant trichomes were hyper-branched, whereas others were hypo-branched, distorted, or clustered. Further, we found that in addition to specifically activating R3 MYB transcription factor TRIPTYCHON (TRY) to modulate trichome specification, TTG2 also integrated cytoskeletal signaling to regulate trichome morphogenesis. The ttg2-6 trichomes displayed aberrant cortical microtubules (cMTs) and actin filaments (F-actin) configurations. Moreover, genetic and biochemical analyses showed that TTG2 could directly bind to the promoter and regulate the expression of BRICK1 (BRK1), which encodes a subunit of the actin nucleation promoting complex suppressor of cyclic AMP repressor (SCAR)/Wiskott-Aldrich syndrome protein family verprolin homologous protein (WAVE). Collectively, taking advantage of ttg2-6, we uncovered a function for TTG2 in facilitating cMTs and F-actin cytoskeleton-dependent trichome development, providing insight into cellular signaling events downstream of the core transcriptional regulation during trichome development in Arabidopsis.

Coordinated inositide lipid-phosphatase activities of synaptojanin modulates actin cytoskeleton organization.

Synaptojanin proteins are evolutionarily conserved regulators of vesicle transport and membrane homeostasis. Disruption of synaptojanin function has been implicated in a wide range of neurological disorders. Synaptojanins act as dual-functional lipid phosphatases capable of hydrolyzing a variety of phosphoinositides (PIPs) through autonomous SAC1-like PIP 4-phosphatase and PIP2 5-phosphatase domains. The rarity of an evolutionary configuration of tethering two distinct enzyme activities in a single protein prompted us to investigate their individual and combined roles in budding yeast. Both PIP and PIP2 phosphatase activities are encoded by multiple gene products and are independently essential for cell viability. In contrast, we observed that the synaptojanin proteins utilized both lipid-phosphatase activities to properly coordinate polarized distribution of actin during the cell cycle. Expression of each activity untethered (in trans) failed to properly reconstitute the basal actin regulatory activity; whereas tethering (in cis), even through synthetic linkers, was sufficient to complement these defects. Studies of chimeric proteins harboring PIP and PIP2 phosphatase domains from a variety of gene products indicate synaptojanin proteins have highly specialized activities and that the length of the linker between the lipid-phosphatase domains is critical for actin regulatory activity. Our data are consistent with synaptojanin possessing a strict requirement for both two-domain configuration for some but not all functions and provide mechanistic insights into a coordinated role of tethering distinct lipid-phosphatase activities.

Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.

A nemaline myopathy-linked mutation inhibits the actin-regulatory functions of tropomodulin and leiomodin.

Actin is a highly expressed protein in eukaryotic cells and is essential for numerous cellular processes. In particular, efficient striated muscle contraction is dependent upon the precise regulation of actin-based thin filament structure and function. Alterations in the lengths of actin-thin filaments can lead to the development of myopathies. Leiomodins and tropomodulins are members of an actin-binding protein family that fine-tune thin filament lengths, and their dysfunction is implicated in muscle diseases. An Lmod3 mutation [G326R] was previously identified in patients with nemaline myopathy (NM), a severe skeletal muscle disorder; this residue is conserved among Lmod and Tmod isoforms and resides within their homologous leucine-rich repeat (LRR) domain. We mutated this glycine to arginine in Lmod and Tmod to determine the physiological function of this residue and domain. This G-to-R substitution disrupts Lmod and Tmod's LRR domain structure, altering their binding interface with actin and destroying their abilities to regulate thin filament lengths. Additionally, this mutation renders Lmod3 nonfunctional in vivo. We found that one single amino acid is essential for folding of Lmod and Tmod LRR domains, and thus is essential for the opposing actin-regulatory functions of Lmod (filament elongation) and Tmod (filament shortening), revealing a mechanism underlying the development of NM.

Molecular dynamics simulations support a multistep pathway for activation of branched actin filament nucleation by Arp2/3 complex.

Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of ∼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.

A deep learning framework for quantitative analysis of actin microridges.

Microridges are evolutionarily conserved actin-rich protrusions present on the apical surface of squamous epithelial cells. In zebrafish epidermal cells, microridges form self-evolving patterns due to the underlying actomyosin network dynamics. However, their morphological and dynamic characteristics have remained poorly understood owing to a lack of computational methods. We achieved ~95% pixel-level accuracy with a deep learning microridge segmentation strategy enabling quantitative insights into their bio-physical-mechanical characteristics. From the segmented images, we estimated an effective microridge persistence length of ~6.1 µm. We discovered the presence of mechanical fluctuations and found relatively greater stresses stored within patterns of yolk than flank, indicating distinct regulation of their actomyosin networks. Furthermore, spontaneous formations and positional fluctuations of actin clusters within microridges were associated with pattern rearrangements over short length/time-scales. Our framework allows large-scale spatiotemporal analysis of microridges during epithelial development and probing of their responses to chemical and genetic perturbations to unravel the underlying patterning mechanisms.

Regulation of the Scar/WAVE complex in migrating cells: A summary of our understanding.

Eukaryotic cell migration requires continuous supply of actin polymers at the leading edges to make and extend lamellipodia or pseudopodia. Linear and branched filamentous actin polymers fuel cell migration. Branching of actin polymers in the lamellipodia/pseudopodia is facilitated by the actin-related protein (Arp) 2/3 complex, whose function is essentially controlled by the Scar/WAVE complex. In cells, the Scar/WAVE complex remains inactive, and its activation is a highly regulated and complex process. In response to signalling cues, GTP-bound Rac1 associates with Scar/WAVE and causes activation of the complex. Rac1 is essential but not sufficient for the activation of the Scar/ WAVE complex, and it requires multiple regulators, such as protein interactors and modifications (phosphorylation, ubiquitylation, etc.). Although our understanding of the regulation of the Scar/WAVE complex has improved over the last decade, it remains enigmatic. In this review, we have provided an overview of actin polymerization and discussed the importance of various regulators of Scar/WAVE activation.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Mechanical stretching induces fibroblasts apoptosis through activating Piezo1 and then destroying actin cytoskeleton.

The anatomical positions of pelvic floor organs are maintained by ligaments and muscles. Stress urinary incontinence (SUI) occurs when the pelvic floor tissues are repeatedly stimulated by excessive mechanical tension that exceeds the bearing capacity of ligaments or muscles. Besides, cells respond mechanically to mechanical stimulation by reconstituting the Piezo1 and cytoskeletal system. The aim of this study is to determine how Piezo1 and actin cytoskeleton are involved in the mechanized stretch (MS) induced apoptosis of human anterior vaginal wall fibroblasts (hAVWFs) and the mechanism. A four-point bending device was used to provide mechanical stretching to establish a cellular mechanical damage model. The apoptosis of hAVWFs cells in non-SUI patients was significantly increased by MS, which exhibited apoptosis rates comparable to those of SUI patients. Based on these findings, Piezo1 connects the actin cytoskeleton to the apoptosis of hAVWFs cells, providing an idea for the clinical diagnosis and treatment of SUI. However, the disassembly of the actin cytoskeleton suppressed the protective effect of Piezo1 silencing on MS. Based on these findings, Piezo1 connects the actin cytoskeleton to apoptosis of hAVWFs, providing new insight for the clinical diagnosis and treatment of SUI.

Actin Depolymerization Factor ADF1 Regulated by MYB30 Plays an Important Role in Plant Thermal Adaptation.

Actin filaments are essential for plant adaptation to high temperatures. However, the molecular mechanisms of actin filaments in plant thermal adaptation remain unclear. Here, we found that the expression of Arabidopsis actin depolymerization factor 1 (AtADF1) was repressed by high temperatures. Compared with wild-type seedlings (WT), the mutation of AtADF1 and the overexpression of AtADF1 led to promoted and inhibited plant growth under high temperature conditions, respectively. Further, high temperatures induced the stability of actin filaments in plants. Compared with WT, Atadf1-1 mutant seedlings showed more stability of actin filaments under normal and high temperature conditions, while the AtADF1 overexpression seedlings showed the opposite results. Additionally, AtMYB30 directly bound to the promoter of AtADF1 at a known AtMYB30 binding site, AACAAAC, and promoted the transcription of AtADF1 under high temperature treatments. Genetic analysis further indicated that AtMYB30 regulated AtADF1 under high temperature treatments. Chinese cabbage ADF1 (BrADF1) was highly homologous with AtADF1. The expression of BrADF1 was inhibited by high temperatures. BrADF1 overexpression inhibited plant growth and reduced the percentage of actin cable and the average length of actin filaments in Arabidopsis, which were similar to those of AtADF1 overexpression seedlings. AtADF1 and BrADF1 also affected the expression of some key heat response genes. In conclusion, our results indicate that ADF1 plays an important role in plant thermal adaptation by blocking the high-temperature-induced stability of actin filaments and is directly regulated by MYB30.

Morphological signatures of actin organization in single cells accurately classify genetic perturbations using CNNs with transfer learning.

The actin cytoskeleton plays essential roles in countless cell processes, from cell division to migration to signaling. In cancer cells, cytoskeletal dynamics, cytoskeletal filament organization, and overall cell morphology are known to be altered substantially. We hypothesize that actin fiber organization and cell shape may carry specific signatures of genetic or signaling perturbations. We used convolutional neural networks (CNNs) on a small fluorescence microscopy image dataset of retinal pigment epithelial (RPE) cells and triple-negative breast cancer (TNBC) cells for identifying morphological signatures in cancer cells. Using a transfer learning approach, CNNs could be trained to accurately distinguish between normal and oncogenically transformed RPE cells with an accuracy of about 95% or better at the single cell level. Furthermore, CNNs could distinguish transformed cell lines differing by an oncogenic mutation from each other and could also detect knockdown of cofilin in TNBC cells, indicating that each single oncogenic mutation or cytoskeletal perturbation produces a unique signature in actin morphology. Application of the Local Interpretable Model-Agnostic Explanations (LIME) method for visually interpreting the CNN results revealed features of the global actin structure relevant for some cells and classification tasks. Interestingly, many of these features were supported by previous biological observation. Actin fiber organization is thus a sensitive marker for cell identity, and identification of its perturbations could be very useful for assaying cell phenotypes, including disease states.

Actin depolymerizing factor ADF7 inhibits actin bundling protein VILLIN1 to regulate root hair formation in response to osmotic stress in Arabidopsis.

Actin cytoskeleton is essential for root hair formation. However, the underlying molecular mechanisms of actin dynamics in root hair formation in response to abiotic stress are largely undiscovered. Here, genetic analysis showed that actin-depolymerizing protein ADF7 and actin-bundling protein VILLIN1 (VLN1) were positively and negatively involved in root hair formation of Arabidopsis respectively. Moreover, RT-qPCR, GUS staining, western blotting, and genetic analysis revealed that ADF7 played an important role in inhibiting the expression and function of VLN1 during root hair formation. Filament actin (F-actin) dynamics observation and actin pharmacological experiments indicated that ADF7-inhibited-VLN1 pathway led to the decline of F-actin bundling and thick bundle formation, as well as the increase of F-actin depolymerization and turnover to promote root hair formation. Furthermore, the F-actin dynamics mediated by ADF7-inhibited-VLN1 pathway was associated with the reactive oxygen species (ROS) accumulation in root hair formation. Finally, ADF7-inhibited-VLN1 pathway was critical for osmotic stress-induced root hair formation. Our work demonstrates that ADF7 inhibits VLN1 to regulate F-actin dynamics in root hair formation in response to osmotic stress, providing the novel evidence on the F-actin dynamics and their molecular mechanisms in root hair formation and in abiotic stress.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization.

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Tools for studying the cytoskeleton during plant cell division.

The plant cytoskeleton regulates fundamental biological processes, including cell division. How to experimentally perturb the cytoskeleton is a key question if one wants to understand the role of both actin filaments (AFs) and microtubules (MTs) in a given biological process. While a myriad of mutants are available, knock-out in cytoskeleton regulators, when nonlethal, often produce little or no phenotypic perturbation because such regulators are often part of a large family, leading to functional redundancy. In this review, alternative techniques to modify the plant cytoskeleton during plant cell division are outlined. The different pharmacological and genetic approaches already developed in cell culture, transient assays, or in whole organisms are presented. Perspectives on the use of optogenetics to perturb the plant cytoskeleton are also discussed.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Profilin-1 negatively controls osteoclast migration by suppressing the protrusive structures based on branched actin filaments.

BACKGROUND: Profilin-1 (Pfn1), an evolutionarily conserved actin-binding protein, is an important regulator of the cytoskeleton. We previously reported the osteoclast-specific Pfn1-conditional knockout (cKO) mice had postnatal osteolytic phenotype with craniofacial and long-bone deformities associated with increased migration of cultured osteoclasts. We hypothesized the increased cellular processes structured with branched actin filaments may underlies the mechanism of increased bone resorption in these mutant mice. MATERIALS AND METHODS: The morphological structure and cell migration of the cultured osteoclasts were analyzed using fluorescent microscopy and time-lapse image capturing. Fractional migration distances, as well as the index of protrusive structures (%-PB) that evaluates relative border length of the protrusion were compared between the cells from control and Pfn1-cKO mice. RESULTS: Time-lapse image analysis showed that %-PB was significantly larger in Pfn1-cKO osteoclasts. In addition, the fractional migration distance was positively correlated with the index. When the branched actin filament organization was suppressed by chemical inhibitors, the osteoclast migration was declined. Importantly, the suppression was more extensive in Pfn1-cKO than in control osteoclasts. CONCLUSION: Our results indicated the causative involvement of the increased branched actin filament formation at least in part for their excessive migration. Our findings provide a mechanistic rationale for testing novel therapeutic approaches targeting branched actin filaments in osteolytic disorders.