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Preeclampsia (PE) is a hypertensive disease associated with increased endothelial cell dysfunction caused by systemic oxidative stress. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that are upregulated in several types of cancer. However, its role in PE remains unclear. In this study, we found that ACTN4 was localized in placenta vascular endothelial cells (ECs), and its expression was downregulated in primary human umbilical vein endothelial cells (HUVECs) from severe preeclamptic patients compared to that in HUVECs from normotensive pregnant women. ACTN4 expression was also decreased in normotensive HUVECs treated with H 2 O 2 . Downregulation of ACTN4 by siRNA or H 2 O 2 treatment promoted normotensive HUVEC apoptosis and increased p38-MAPK phosphorylation along with elevated levels of p53 phosphorylation, caspase cascade proteins, and bax and repressed expression of bcl-2. Conversely, upregulation of ACTN4 in PE HUVECs significantly inhibited apoptosis and decreased p38-MAPK phosphorylation compared to that of the PE HUVEC controls. In addition, overexpression of ACTN4 in normotensive HUVECs attenuated H 2 O 2 treatment-induced apoptosis with decreased p53 phosphorylation, caspase cascade, and bax expression levels and increased expression of bcl-2 compared to that of only H 2 O 2 treatment. Moreover, the suppression of ACTN4 induced apoptosis, which could be blocked by the p38-MAPK inhibitor SB202190. Collectively, these results demonstrate that dysregulated ACTN4 expression may be associated with PE due to its effects on endothelial cell apoptosis via the p38-MAPK/p53 apoptosis pathway.

Objective: The special status accorded to elite athletes stems from their uncommon genetic potential to excel in world-class power sports (PS). Genetic polymorphisms have been reported to influence elite PS status. Reports of associations between the α-actinin-3 gene (ACTN3) R577X polymorphism and PS have been inconsistent. In light of new published studies, we perform a meta-analysis to further explore the roles of this polymorphism in PS performance among elite athletes., Methods: Multi-database literature search yielded 44 studies from 38 articles. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were used in estimating associations (significance threshold was set at Pa ≤ 0.05) using the allele-genotype model (R and X alleles, RX genotype). Outlier analysis was used to examine its impact on association and heterogeneity outcomes. Subgroup analysis was race (Western and Asian) and gender (male/female)-based. Interaction tests were applied to differential outcomes between the subgroups, P-values of which were Bonferroni corrected (Pinteraction BC). Tests for sensitivity and publication bias were performed., Results: Significant overall R allele effects (OR 1.21, 95% CI 1.07-1.37, Pa = 0.002) were confirmed in the Western subgroup (OR 1.11, 95% CI 1.01-1.22, Pa = 0.02) and with outlier treatment (ORs 1.12-1.20, 95% CIs 1.02-1.30, Pa < 10-5-0.01). This treatment resulted in acquired significance of the RX effect in Asian athletes (OR 1.91, 95% CI 1.25-2.92, Pa = 0.003). Gender analysis dichotomized the RX genotype and R allele effects as significantly higher in male (OR 1.14, 95% CI 1.02-1.28, Pa = 0.02) and female (OR 1.58, 95% CI 1.21-2.06, Pa = 0.0009) athletes, respectively, when compared with controls. Significant R female association was improved with a test of interaction (Pinteraction BC = 0.03). The overall, Asian and female outcomes were robust. The R allele results were more robust than the RX genotype outcomes. No evidence of publication bias was found., Conclusions: In this meta-analysis, we present clear associations between the R allele/RX genotype in the ACTN3 polymorphism and elite power athlete status. Significant effects of the R allele (overall analysis, Western and female subgroups) and RX genotype (Asians and males) were for the most part, results of outlier treatment. Interaction analysis improved the female outcome. These robust findings were free of publication bias., Competing Interests: The authors have declared that no competing interests exist.

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells' lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Acquired radioresistance accompanied with increased metastatic potential is a major hurdle in effective radiotherapy of breast cancers. However, the nature of their inter-dependence and the underlying mechanism remains largely intangible. By employing radioresistant (RR) cell lines, we herein demonstrate that MCF-7 RR cells display phenotypic and molecular alterations evocative of epithelial to mesenchymal transition (EMT) with increased traction forces and membrane ruffling culminating in boosted invasiveness. We then show that these changes can be attributed to overexpression of alpha-actinin-4 (ACTN4), with ACTN4 knockdown near-completely abrogating both radioresistance and EMT-associated changes. We further found that in MCF-7 RR cells, ACTN4 mediates the observed effects by activating AKT, and downstream AKT/GSK3β signalling. Though ACTN4 plays a similar role in mediating radioresistance and invasiveness in MDA-MB-231 RR cells, co-immunoprecipitation studies reveal that these changes are effected through increased association with AKT and not by overexpression of AKT. Taken together, our study identifies ACTN4/AKT/GSK3β as a novel pathway regulating radioresistance coupled invasion which can be further explored to improve the radiotherapeutic gain., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2 (Tvα-actinin 2) has been used to diagnose trichomoniasis. Tvα-actinin 2 was dissected into three parts; the N-terminal, central, and C-terminal portions of the protein (#1, #2, and #3, respectively). Western blot of these Tvα-actinin 2 proteins with pooled patients' sera indicated that #2 and #3, but not #1, reacted with those sera. Immunofluorescence assays of two different forms of T. vaginalis (trophozoites and amoeboid forms), using anti-Tvα-actinin 2 antibodies, showed localization of Tvα-actinin 2 close to the plasma membranes of the amoeboid form. Fractionation experiments indicated the presence of Tvα-actinin 2 in cytoplasmic, membrane, and secreted proteins of T. vaginalis . Binding of fluorescence-labeled Trichomonas to vaginal epithelial cells and prostate cells was decreased in the antibody blocking experiment using anti-Tvα-actinin 2 antibodies. Pretreatment of T. vaginalis with anti-rTvα-actinin 2 antibodies also resulted in reduction in its cytotoxicity. Flow cytometry, ligand-binding immunoblotting assay, and observation by fluorescence microscopy were used to detect the binding of recombinant Tvα-actinin 2 to human epithelial cell lines. Specifically, the truncated N-terminal portion of Tvα-actinin 2, Tvα-actinin 2 #1, was shown to bind directly to vaginal epithelial cells. These data suggest that α-actinin 2 is one of the virulence factors responsible for the pathogenesis of T. vaginalis by serving as an adhesin to the host cells.

Key Points: Ion channels are transmembrane proteins that are synthesized within the cells but need to be trafficked to the cell membrane for the channels to function. Small-conductance, Ca 2+ -activated K + channels (SK, K Ca 2) are unique subclasses of K + channels that are regulated by Ca 2+ inside the cells; they are expressed in human atrial myocytes and responsible for shaping atrial action potentials. We have previously shown that interacting proteins of SK2 channels are important for channel trafficking to the membrane. Using total internal reflection fluorescence (TIRF) and confocal microscopy, we studied the mechanisms by which the surface membrane localization of SK2 (K Ca 2.2) channels is regulated by their interacting proteins. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes., Abstract: The normal function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane. Small-conductance, Ca 2+ -activated K + channels (SK, K Ca 2) are expressed in human atrial myocytes and are responsible for shaping atrial action potentials. Understanding the mechanisms of SK channel trafficking may provide new insights into the regulation controlling the repolarization of atrial myocytes. We have previously demonstrated that the C- and N-termini of SK2 channels interact with the actin-binding proteins α-actinin2 and filamin A, respectively. However, the roles of the interacting proteins on SK2 channel trafficking remain incompletely understood. Using total internal reflection fluorescence (TIRF) microscopy, we studied the mechanisms of surface membrane localization of SK2 (K Ca 2.2) channels. When SK2 channels were co-expressed with filamin A or α-actinin2, the membrane fluorescence intensity of SK2 channels increased significantly. We next tested the effects of primaquine and dynasore on SK2 channels expression. Treatment with primaquine significantly reduced the membrane expression of SK2 channels. In contrast, treatment with dynasore failed to alter the surface membrane expression of SK2 channels. Further investigations using constitutively active or dominant-negative forms of Rab GTPases provided additional insights into the distinct roles of the two cytoskeletal proteins on the recycling processes of SK2 channels from endosomes. α-Actinin2 facilitated recycling of SK2 channels from both early and recycling endosomes while filamin A probably aids the recycling of SK2 channels from recycling endosomes., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

During spreading and migration, the leading edges of cells undergo periodic protrusion-retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion-retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation., (© 2016 Meacci, Wolfenson, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Purpose: To examine the effect of α-actinin-3 deficiency due to homozygosity for the ACTN3 577X-allele on contractile and morphological properties of fast muscle fibers in non-athletic young men., Methods: A biopsy was taken from the vastus lateralis of 4 RR and 4 XX individuals to test for differences in morphologic and contractile properties of single muscle fibers. The cross-sectional area of the fiber and muscle fiber composition was determined using standard immunohistochemistry analyses. Skinned single muscle fibers were subjected to active tests to determine peak normalized force (P0), maximal unloading velocity (V0) and peak power. A passive stretch test was performed to calculate Young's Modulus and hysteresis to assess fiber visco-elasticity., Results: No differences were found in muscle fiber composition. The cross-sectional area of type IIa and IIx fibers was larger in RR compared to XX individuals (P<0.001). P0 was similar in both groups over all fiber types. A higher V0 was observed in type IIa fibers of RR genotypes (P<0.001) but not in type I fibers. The visco-elasticity as determined by Young's Modulus and hysteresis was unaffected by fiber type or genotype., Conclusion: The greater V0 and the larger fast fiber CSA in RR compared to XX genotypes likely contribute to enhanced whole muscle performance during high velocity contractions.

The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing.

Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease.

Interaction of cells with extracellular matrix (ECM) regulates cell shape, differentiation and polarity. This effect has been widely observed in cells grown on substrates with various patterned features, stiffness and surface chemistry. It has been postulated that mechanical confinement of cells by the substrate causes a redistribution of tension in cytoskeletal proteins resulting in cytoskeletal reorganization through force sensitive pathways. However, the mechanisms for force transduction during reorganization remain unclear. In this study, using FRET based force sensors we have measured tension in an actin cross-linking protein, α-actinin, and followed reorganization of actin cytoskeleton in real time in HEK cells grown on patterned substrates. We show that the patterned substrates cause a redistribution of tension in α-actinin that coincides with cytoskeleton reorganization. Higher tension was observed in portions of cells where they form bridges across inhibited regions of the patterned substrates; the attachment to the substrate is found to release tension. Real time measurements of α-actinin tension and F-actin arrangement show that an increase in tension coincides with formation of F-actin bundles at the cell periphery during cell-spreading across inhibited regions, suggesting that mechanical forces stimulate cytoskeleton enhancement. Rho-ROCK inhibitor (Y27632) causes reduction in actinin tension followed by retraction of bridged regions. Our results demonstrate that changes in cell shape and expansion over patterned surfaces is a force sensitive process that requires actomyosin contractile force involving Rho-ROCK pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.

α-Actinin-3 is a Z-disc protein expressed only in type II muscle fibers. A polymorphism in the ACTN3 gene (R577X) results in lack of α-actinin-3 in XX genotype. The prevalence of the mutated X-allele is lower among power/sprint oriented athletes compared with controls, indicating that the lack of α-actinin-3 is detrimental in these sports, but a mechanistic link has not been established. Results from Actn3-knockout (KO) mouse model suggest that α-actinin-3 may affect muscle mass and muscle glycogen levels. In the present investigation we examined muscle fiber type composition, cross-sectional fiber area (CSA), and muscle glycogen levels at baseline in 143 human subjects with different ACTN3 genotypes. In addition, hypertrophy signaling and glycogen utilization in response to sprint exercise were studied in a subset of subjects. Glycogen utilization was analyzed in separate pools of type I and type II fibers. No differences in fiber type composition, CSA, or muscle glycogen levels were observed at baseline across the ACTN3 genotypes. However, the sprint exercise-induced increase in phosphorylation of mTOR and p70S6k was smaller in XX than in RR+RX (P = 0.03 and P = 0.01, respectively), indicating a less pronounced activation of hypertrophy signaling in XX. Glycogen utilization during sprint exercise varied across ACTN3 genotypes in type II fibers (P = 0.03) but not in type I fibers (P = 0.38). The present results are in accordance with findings from the KO mice and reinforce the hypothesis that ACTN3 genotype-associated differences in muscle mass and glycogen utilization provide a mechanistic explanation for the modulation of human performance by the ACTN3 genotype.

α-Actinins are a major class of actin filament cross-linking proteins expressed in virtually all cells. In muscle, actinins cross-link thin filaments from adjacent sarcomeres. In non-muscle cells, different actinin isoforms play analogous roles in cross-linking actin filaments and anchoring them to structures such as cell-cell and cell-matrix junctions. Although actinins have long been known to play roles in cytokinesis, cell adhesion and cell migration, recent studies have provided further mechanistic insights into these functions. Roles for actinins in synaptic plasticity and membrane trafficking events have emerged more recently, as has a 'non-canonical' function for actinins in transcriptional regulation in the nucleus. In the present paper we review recent advances in our understanding of these diverse cell biological functions of actinins in non-muscle cells, as well as their roles in cancer and in genetic disorders affecting platelet and kidney physiology. We also make two proposals with regard to the actinin nomenclature. First, we argue that naming actinin isoforms according to their expression patterns is problematic and we suggest a more precise nomenclature system. Secondly, we suggest that the α in α-actinin is superfluous and can be omitted.

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

In polarized, migrating cells, stress fibers are a highly dynamic network of contractile acto-myosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as α-actinins. As such, α-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, α-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of α-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle α-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of α-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An α-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an α-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of α-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of α-actinin1 at Y12 and α-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

During wound repair, epidermal cells at the edge of an injury establish front-rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin-4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin-4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin-4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin-332 matrix. In actinin-4-knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6β4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin-4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.

MDM2 binding protein (MTBP) is a protein that interacts with oncoprotein murine double minute (MDM2), a major inhibitor of the tumor suppressor p53. Overexpression of MTBP leads to p53-independent cell proliferation arrest, which is in turn blocked by simultaneous overexpression of MDM2. Importantly, reduced expression of MTBP in mice increases tumor metastasis and enhances migratory potential of mouse embryonic fibroblasts regardless of the presence of p53. Clinically, loss of MTBP expression in head and neck squamous cell carcinoma is associated with reduced patient survival, and is shown to serve as an independent prognostic factor when p53 is mutated in tumors. These results indicate the involvement of MTBP in suppressing tumor progression. Our recent findings demonstrate that overexpression of MTBP in human osteosarcoma cells lacking wild-type p53 inhibits metastasis, but not primary tumor growth, when cells are transplanted in femurs of immunocompromised mice. These data indicate that MTBP functions as a metastasis suppressor independent of p53 status. Furthermore, overexpression of MTBP suppresses cell migration and filopodia formation, in part, by inhibiting function of an actin crosslinking protein α-actinin-4. Thus, increasing evidence indicates the significance of MTBP in tumor progression. We summarize published results related to MTBP function and discuss caveats and future directions in this review article.

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell-cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin-Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell-cell adhesive contacts.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3(-/-) (KO) mouse suggest a shift towards 'slow' metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an 'equal environment condition' what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier's biopsy was the only one that presented type I fibers with a complete lack of type II(x) fibers. Properties of hybrid type II(a)/II(x) fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II(a)/II(x) fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm(2)) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II(x) fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.

Background: African-Americans (AAs) are predisposed to non-diabetic (non-DM) end-stage renal disease (ESRD), and studies have shown a genetic component to this risk. Rare mutations in ACTN4 (α-actinin-4), an actin-binding protein expressed in podocytes, cause familial focal segmental glomerulosclerosis., Methods: We assessed the contribution of coding variants in ACTN4 to non-DM ESRD risk in AAs. Nineteen exons, 2,800 bases of the promoter and 392 bases of the 3' untranslated region of ACTN4 were sequenced in 96 AA non-DM ESRD cases and 96 non-nephropathy controls (384 chromosomes). Sixty-seven single-nucleotide polymorphisms (SNPs) including 51 novel SNPs were identified. The SNPs comprised 33 intronic, 21 promoter, 12 exonic, and one 3' variant. Sixty-two of the SNPs were genotyped in 296 AA non-DM ESRD cases and 358 non-nephropathy controls., Results: One SNP, rs10404257, was associated with non-DM ESRD (p < 1.0E-4, odds ratio, OR = 0.76; confidence interval, CI = 0.59-0.98; additive model). Forty-seven SNPs had minor allele frequencies <5%. These SNPs were segregated into risk and protective SNPs, and each category was collapsed into a single marker, designated by the presence or absence of any rare allele. The presence of any rare allele at a risk SNP was significantly associated with non-DM ESRD (p = 0.001, dominant model). The SNPs with the strongest evidence for association (n = 20) were genotyped in an independent set of 467 non-DM ESRD cases and 279 controls. Although rs10404257 was not associated in this replication sample, when the samples were combined, rs10404257 was modestly associated (p = 0.032, OR = 0.78, CI = 0.63-0.98; dominant model). SNPs were tested for interaction with markers in the APOL1 gene, previously associated with non-DM ESRD in AAs, and rs10404257 was modestly associated (p = 0.0261, additive model)., Conclusions: This detailed evaluation of ACTN4 variation revealed limited evidence of association with non-DM ESRD in AAs., (Copyright © 2012 S. Karger AG, Basel.)

The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ∼60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.

Absence of α-actinin-3, encoded by the ACTN3 "speed gene," is associated with poorer sprinting performance in athletes and a slowing of relaxation in fast-twitch muscles of Actn3 knockout (KO) mice. Our first aim was to investigate, at the individual-fiber level, possible mechanisms for this slowed relaxation. Our second aim was to characterize the contractile properties of whole extensor digitorum longus (EDL) muscles from KO mice by age and gender. We examined caffeine-induced Ca(2+) release in mechanically skinned EDL fibers from KO mice, and measured isolated whole EDL contractile properties. The sarcoplasmic reticulum of KO muscle fibers loaded Ca(2+) more slowly than that of wild-types (WTs). Whole KO EDL muscles had longer twitch and tetanus relaxation times than WTs, and reduced mass and cross-sectional area. These effects occurred in both male and female mice, but they diminished with age. These changes in KO muscles and fibers help to explain the effects of α-actinin-3 deficiency observed in athletes., (Copyright © 2010 Wiley Periodicals, Inc.)

Background: Falls among elderly people is a major issue in public health, causing debilitating outcomes including fracture. The identification of genetic risk factors for falling may provide a strategy for effectively targeting falls prevention programs. We investigated whether a common functional variant of skeletal muscle α-actinin-3 (ACTN3 p. R577X) previously associated with impairments in muscle strength, power, and physical functioning represents a risk factor for falls., Methods: Case-control analysis was conducted using two large cohorts of Caucasian postmenopausal women--the North of Scotland Osteoporosis Study (n = 1,245) and the Aberdeen Prospective Osteoporosis Screening Study (n = 2,918)--for whom self-reported falls status and DNA samples were available. Cross-sectional analysis of fallers versus nonfallers at baseline and follow-up was performed. In addition, individuals who reported having fallen at more than one timepoint (recurrent fallers) were compared with those who reported not falling at any timepoint., Results: Association between R577X genotype and falls was identified and validated. Carriage of 577X (one or two copies) was significantly associated with a 33% (10%-61%) increased risk of falling, with the effect apparent at both baseline and follow-up assessments (meta-analysis p = .003 and p = .02, respectively). No significant effect on recurrent falls was observed., Conclusion: This study reports for the first time that the functional ACTN3 R577X genotype represents a genetic risk factor for falling in older females.

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A common polymorphism (R577X) in the ACTN3 gene results in complete deficiency of alpha-actinin-3 protein in approximately 16% of humans worldwide. The presence of alpha-actinin-3 protein is associated with improved sprint/power performance in athletes and the general population. Despite this, there is evidence that the null genotype XX has been acted on by recent positive selection, likely due to its emerging role in the regulation of muscle metabolism. alpha-Actinin-3 deficiency reduces the activity of glycogen phosphorylase and results in a fundamental shift toward more oxidative pathways of energy utilization.

Biological sensors and their ability to detect and respond to change in the cellular environment can be modulated by protein scaffolds acting within their interaction network. The skeletal muscle alpha-actinins have been considered as primarily structural scaffold proteins. However, deficiency of alpha-actinin-3 due to a common null polymorphism results in predominantly metabolic changes in skeletal muscle function. In this review, we explore the range of phenotypes associated with alpha-actinin-3 deficiency, and draw supporting evidence from known interaction partners for its role as a scaffold which acts to modulate biological sensors that result in changes in muscle metabolism and structure., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Alpha-actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle alpha-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as alpha-actinin 1 was higher in astrocytomas compared to normal brains whereas alpha-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of alpha-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While alpha-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, alpha-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of alpha-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, alpha-actinin 1 and 4 had contrasted biochemical properties as alpha-actinin 4 was significantly more abundant in the actin cytoskeleton than alpha-actinin 1. Collectively, these findings suggest that alpha-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells., (Published by Elsevier Inc.)

Objectives: To examine actinin-4 expression levels in bladder cancer, in particular its levels during cellular growth and invasion. Actinin-4 is an actin-binding protein that is associated with cell motility and cancer metastasis., Methods: Relative messenger ribonucleic acid (mRNA) and protein expression of actinin-4 in normal bladder and bladder cancer cell lines was determined by quantitative real-time polymerase chain reaction and Western blot analysis. Actinin-4 expression was also localized in bladder cancer cells and tissues using immunohistochemistry. The growth and invasion activity of bladder cancer cells was evaluated using cell growth and in vitro cell invasion assays, and compared with that of bladder cancer cells treated with actinin-4 small interfering ribonucleic acids., Results: Actinin-4 mRNA and protein levels were elevated in bladder cancer cells that are known to exhibit increased growth and invasion activity. Protein expression was predominantly observed in the cytoplasm of the invasive bladder cancer cells and tissues. Treatment of bladder cancer cell lines with actinin-4 small interfering ribonucleic acids suppressed the invasive potential of the cells, but did not alter their growth., Conclusions: The current study demonstrates that actinin-4 mRNA and protein levels are elevated in bladder cancer cells lines that exhibit increased growth and invasion activity. In addition, actinin-4 knockdown inhibited invasion of bladder cancer cells, but did not alter their growth. In conclusion, we hypothesize that the accumulation of actinin-4 in the cell cytoplasm is related to an increased susceptibility of tumor invasion and metastasis., (2010 Elsevier Inc. All rights reserved.)

In humans, there are two skeletal muscle alpha-actinins, encoded by ACTN2 and ACTN3, and the ACTN3 genotype is associated with human athletic performance. Remarkably, approximately 1 billion people worldwide are deficient in alpha-actinin-3 due to the common ACTN3 R577X polymorphism. The alpha-actinins are an ancient family of actin-binding proteins with structural, signalling and metabolic functions. The skeletal muscle alpha-actinins diverged approximately 250-300 million years ago, and ACTN3 has since developed restricted expression in fast muscle fibres. Despite ACTN2 and ACTN3 retaining considerable sequence similarity, it is likely that following duplication there was a divergence in function explaining why alpha-actinin-2 cannot completely compensate for the absence of alpha-actinin-3. This paper focuses on the role of skeletal muscle alpha-actinins, and how possible changes in functions between these duplicates fit in the context of gene duplication paradigms.

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that alpha-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an alpha-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for alpha-actinin-1 and -4. Co-expressing alpha-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing alpha-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that alpha-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of alpha-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of alpha-actinin determine the Z-disc width: fast fibres have narrow (approximately 30-50 nm) Z-discs and slow and cardiac fibres have wide (approximately 100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.

The human sarcomeric alpha-actinins (ACTN2 and ACTN3) are major structural components of the Z line in skeletal muscle; they play a role in the maintenance of sarcomeric integrity and also interact with a wide variety of structural, signaling and metabolic proteins. ACTN2 is expressed in all muscle fibers, and expression of ACTN3 is restricted to the type 2 (fast glycolytic) fibers that are responsible for forceful contraction at high velocity. There is a common stop codon polymorphism R577X in the ACTN3 gene. Homozygosity for the R577X null-allele results in the absence of alpha-actinin-3 in fast muscle fibers with frequencies that vary from < 1% in Africans to approximately 18% in Caucasians. A number of association studies have demonstrated that the ACTN3 R577X genotype influences athletic performance in Caucasians; the frequency of the XX genotype is significantly lower than controls in sprint athletes, and it appears that alpha-actinin-3 deficiency is detrimental to sprint performance. In the general population, the ACTN3 genotype contributes to the normal variations in muscle strength and sprinting speed. In an Actn3 knockout mouse model, alpha-actinin-3 deficiency is associated with a shift in the characteristics of fast, glycolytic 2B muscle fibers towards a slow phenotype, with decreased muscle mass and fiber diameter, slower contractile properties, increased fatigue resistance, and an increase in oxidative enzyme activity. The shift towards a more efficient oxidative metabolism may underlie the selective advantage of the X-allele during evolution. In turn, the shift towards a 'slow' muscle phenotype in fast muscle fibers likely explains why loss of alpha-actinin-3 is detrimental to sprint performance., (2009 S. Karger AG, Basel)

We have shown previously that, in astrocytoma cells, synemin is present at the leading edge, an unusual localization for an intermediate filament (IF) protein. Here, we report that synemin down-regulation with specific small hairpin RNAs (shRNAs) sharply decreased the migration of astrocytoma cells. The presence of synemin at the leading edge also correlated with a high migratory potential, as shown by comparing astrocytoma cells to carcinoma cells without synemin at the leading edge. Synemin-silenced astrocytoma cells were smaller and spread more slowly than controls. In addition, synemin silencing reduced proliferation without increasing apoptosis. The adhesion to substratum and distribution of vinculin in focal contacts of synemin-silenced astrocytoma cells were similar to those of controls. Synemin-silenced cells, however, exhibited a reduction in the amount of filamentous (F) -actin and of alpha-actinin, but not of vinculin, associated with F-actin. Altogether, these results demonstrate that synemin is important for the malignant behavior of astrocytoma cells and that it contributes to the high motility of these cells by modulating the dynamics of alpha-actinin and actin.

Alpha-actinins are critical components of the actin cytoskeleton. Here we show that alpha-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). Alpha-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express alpha-actinin-1 and -4, alpha-actinin-4 interacted and colocalized with eNOS. Addition of alpha-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of alpha-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of alpha-actinin-4 by siRNA increased eNOS activity in BAECs. The alpha-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on alpha-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased alpha-actinin-4-eNOS interaction, leading to translocation of alpha-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced alpha-actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with alpha-actinin-4 and calmodulin.

Integrins are alphabeta heterodimeric receptors that mediate attachment of cells to the extracellular matrix and therefore play important roles in cell adhesion, migration, proliferation and survival. Among the cytoskeletal proteins that interact directly with the beta-chain cytoplasmic domain, talin has emerged as playing a critical role in integrin activation and linkage to the actin cytoskeleton. Talin (2541 amino acids) is an elongated (60 nm) flexible antiparallel dimer, with a small globular head connected to an extended rod. The talin head contains a FERM (4.1/ezrin/radixin/moesin) domain (residues 86-400) with binding sites for several beta integrin cytodomains and the talin rod contains a second lower-affinity integrin-binding site, a highly conserved C-terminal actin-binding site and also several binding sites for vinculin. We have determined previously the crystal structures of two domains from the talin rod, spanning residues 482-789. Talin-(482-655), which contains a VBS (vinculin-binding site), folds into a five-helix bundle whereas talin-(656-789) is a four-helix bundle. We have also reported the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. In the present paper, we consider how binding sites buried within the folded helical bundles of talin and alpha-actinin form interactions with vinculin.

Alpha-actinins are major structural components of the Z-discs in skeletal muscle. Alpha-actinin 3 is encoded by the ACTN3 gene and is expressed only in type II muscle fibres. Homozygosity for the nonsense mutation, 577X, within ACTN3 results in deficiency of alpha-actinin-3 but does not result in an abnormal muscular phenotype. Previous research has found an association of the 577R allele with sprinting and/or power performance. It has also been suggested that the 577X allele may confer an advantage during endurance events. Four hundred and fifty seven Caucasian male triathletes who completed either the 2000 and/or 2001 226 km South African Ironman Triathlons, and 143 Caucasian controls, were genotyped for the R577X mutation within the ACTN3 gene. There were no significant differences in either the genotype (P = 0.486) or allele (P = 0.375) frequencies within the fastest, middle of the field or slowest Caucasian male finishers and the control population. In conclusion, the R577X polymorphism within the ACTN3 gene was not associated with ultra-endurance performance in the 2000 and 2001 South African Ironman Triathlons.

Although signal transduction pathways provide spatiotemporal control of cytokinesis, additional regulation likely occurs through complex cytoskeletal network interactions. In this issue of Developmental Cell, Mukhina et al. (2007) show that myosin-II modulates the cortical lifetime of the actin crosslinker alpha-actinin, which in turn tunes actin filament dynamics, thereby controlling furrow ingression.

Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.

More than a billion humans worldwide are predicted to be completely deficient in the fast skeletal muscle fiber protein alpha-actinin-3 owing to homozygosity for a premature stop codon polymorphism, R577X, in the ACTN3 gene. The R577X polymorphism is associated with elite athlete status and human muscle performance, suggesting that alpha-actinin-3 deficiency influences the function of fast muscle fibers. Here we show that loss of alpha-actinin-3 expression in a knockout mouse model results in a shift in muscle metabolism toward the more efficient aerobic pathway and an increase in intrinsic endurance performance. In addition, we demonstrate that the genomic region surrounding the 577X null allele shows low levels of genetic variation and recombination in individuals of European and East Asian descent, consistent with strong, recent positive selection. We propose that the 577X allele has been positively selected in some human populations owing to its effect on skeletal muscle metabolism.

Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.

Mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant human kidney disease. Mice deficient in alpha-actinin-4 develop a recessive phenotype characterized by kidney failure, proteinuria, glomerulosclerosis, and retraction of glomerular podocyte foot processes. However, the mechanism by which alpha-actinin-4 deficiency leads to glomerular disease has not been defined. Here, we examined the effect of alpha-actinin-4 deficiency on the adhesive properties of podocytes in vivo and in a cell culture system. In alpha-actinin-4-deficient mice, we observed a decrease in the number of podocytes per glomerulus compared with wild-type mice as well as the presence of podocyte markers in the urine. Podocyte cell lines generated from alpha-actinin-4-deficient mice were less adherent than wild-type cells to glomerular basement membrane (GBM) components collagen IV and laminin 10 and 11. We also observed markedly reduced adhesion of alpha-actinin-4-deficient podocytes under increasing shear stresses. This adhesion deficit was restored by transfecting cells with alpha-actinin-4-GFP. We tested the strength of the integrin receptor-mediated linkages to the cytoskeleton by applying force to microbeads bound to integrin using magnetic pulling cytometry. Beads bound to alpha-actinin-4-deficient podocytes showed greater displacement in response to an applied force than those bound to wild-type cells. Consistent with integrin-dependent alpha-actinin-4-mediated adhesion, phosphorylation of beta1-integrins on alpha-actinin-4-deficient podocytes is reduced. We rescued the phosphorylation deficit by transfecting alpha-actinin-4 into alpha-actinin-4-deficient podocytes. These results suggest that alpha-actinin-4 interacts with integrins and strengthens the podocyte-GBM interaction thereby stabilizing glomerular architecture and preventing disease.

The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin, a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a later stage. The formation of functional sarcomeres coincided with contractions in normal hearts at stage 35. A-bands and I-bands were formed at stage 35 and did not change at stage 39. The widening of Z-bodies into z-lines was the main developmental difference between stage 35 and 39 normal hearts. Relative to normal hearts, a reduction of sarcomeric protein levels in mutant hearts at stage 35 was found, and a greater reduction occurred at later stages. The lower level of tropomyosin limited the areas where organized myofibrils formed in the mutant. The areas that had tropomyosin staining also had staining for alpha-actinin and myosin. Early myofibrils formed in these areas but the A-bands and I-bands were shorter than normal. At a later stage in the mutant, A-bands and I-bands remained shorter and importantly the Z-bodies also did not form wider z-lines.

The physiological targets regulated by MEF2 in striated muscle are not completely known. Several recent studies have identified novel downstream target genes and shed light on the global transcriptional network regulated by MEF2 in muscle. In our continuing effort to identify novel, downstream pathways controlled by MEF2, we have used mef2a knock-out mice to find those genes dependent on MEF2A transcriptional activity. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the Z-disc/costameric region in striated muscle. This gene, called myomaxin, was identified as a gene markedly down-regulated in MEF2A knock-out hearts. Myomaxin is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy associated gene 3 (CMYA3). Myomaxin is expressed as a single, large transcript of approximately 11 kilobases in adult heart and skeletal muscle with an open reading frame of 3,283 amino acids. The protein encoded by the myomaxin gene is related to the actin-binding protein Xin and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that Myomaxin functions directly downstream of MEF2A at the peripheral Z-disc complex in striated muscle potentially playing a role in regulating cytoarchitectural integrity.

In HER2 (ErbB2)-overexpressing cells, transforming growth factor beta (TGF-beta), via activation of phosphoinositide-3 kinase (PI3K), recruits actin and actinin to HER2, which then colocalizes with Vav2, activated Rac1, and Pak1 at cell protrusions. This results in prolonged Rac1 activation, enhanced motility and invasiveness, Bad phosphorylation, uncoupling of Bad/Bcl-2, and enhanced cell survival. The recruitment of the HER2/Vav2/Rac1/Pak1/actin/actinin complex to lamellipodia was abrogated by actinin siRNAs, dominant-negative (dn) p85, gefitinib, and dn-Rac1 or dn-Pak1, suggesting that the reciprocal interplay of PI3K, HER2 kinase, and Rac GTPases with the actin cytoskeleton is necessary for TGF-beta action in oncogene-overexpressing cells. Thus, by recruiting the actin skeleton, TGF-beta "cross-links" this signaling complex at cell lamellipodia; this prolongs Rac1 activation and increases metastatic properties and survival of HER2-overexpressing cells.