Anchor Actin Filaments Research Articles

Abstract 14355: Mono- and Biallelic Structural Variants in Alpha-actinin 2 Cause Contractile Dysfunction and Cardiomyopathy in Humans

Background and Aim: Cardiomyopathy is a common cause of sudden cardiac death in children that often requires heart transplantation. Alpha-actinins are critical cytoskeletal proteins that anchor actin filaments within the cardiac sarcomere. Mutations in ACTN2 have been associated with cardiomyopathy, but the mechanisms behind how those lead to cardiac dysfunction remain poorly understood. The aim of the present study was to investigate the effects of two novel structural ACTN2 mutations in human cardiac tissue and patient-specific iPSC-derived cardiomyocytes. Methods and Results: We identified a patient homozygous for a stop-gain mutation (p.Q860X) and a family heterozygous for a large exon 8-10 deletion with a 41bp insertion (indel) in ACTN2 , using a custom mutation pipeline optimized for rare variant discovery. In the explanted cardiac tissue of the homozygous patient, we observed mild hypertrophy and interstitial fibrosis. Patient-specific hiPSC-CMs from both families were hypertrophic, displayed sarcomeric structural disarray on transmission electron microscopy, and had slower contractile velocity compared to control hiPSC-CMs. The ACTN2indel protein was expressed, with subsequent incorporation into cardiac sarcomeres. In the homozygous stop-gain cells and normal controls, we used Co-IP followed by mass-spectrometry to identify C-terminal interacting proteins that are disrupted with C terminal loss. We found an intricate ACTN2 interactome with many sarcolemma-associated proteins and showed lack of interaction of the truncated ACTN2 with ACTN1 and GJA1 (Fig. 1). ACTN1 association was verified using colocalization and Co-IP followed by Western blot. Conclusion: We provide evidence that two loss of function genetic variants in ACTN2 are associated with contractile dysfunction and lead to cardiac abnormalities through distinct mechanisms within cardiac sarcomeres and through lack of critical protein-protein interactions.

NOVEL ALPHA-ACTININ 2 MUTATIONS ARE ASSOCIATED WITH CARDIOMYOPATHY AND HYPERTROPHY IN HUMAN CARDIAC TISSUE AND IPSC-DERIVED CARDIOMYOCYTES

In cardiac and skeletal muscle, alpha-actinins are critical cytoskeletal proteins that anchor actin filaments within the sarcomere. Mutations in ACTN2 have been associated with cardiac abnormalities, including arrhythmias, hypertrophic and dilated cardiomyopathies. However, the mechanisms behind how

Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin Cortex.

All cells in all domains of life possess a cytoskeleton that provides mechanical resistance to deformation and general stability to the plasma membrane. Here, we utilize a two-dimensional scaffolding created by actin filaments to convey mechanical support upon relatively fragile planar bilayer membranes (black lipid membranes, BLMs). Robust biomembranes play a critical role in the development of protein nanopore sensor applications and might also prove helpful in ion-channel research. Our investigation utilizes a minimal actin cortex (MAC) that is formed by anchoring actin filaments to lipid membranes via a biotin-streptavidin-biotin bridge. We characterize the joined structure using various modes of optical microscopy, electrophysiology, and applied mechanical stress (including measurements of elastic modulus). Our findings show the resulting structure includes a thin supporting layer of actin. Electrical studies indicate that the integrity of the MAC-bilayer composite remains unchanged over the limits of our tests (i.e., hours to days). The actin filament structure can remain intact for months. Minimalistic layering of the actin support network produces an increase in the apparent elastic modulus of the MAC-derivatized bilayer by >100×, compared to unmodified BLMs. Furthermore, the resistance to applied stress improves with the number of actin layers, which can be cross-linked to arbitrary thicknesses, in principle. The weblike support structure retains the lateral fluidity of the BLM, maintains the high electrical resistance typical of traditional BLMs, enables relatively uninhibited molecular access to the lipid surface from bulk solution, and permits nanopore self-assembly and insertion in the bilayer. These interfacial features are highly desirable for ion-channel and nanopore sensing applications.

Arabidopsis formin 2 regulates cell-to-cell trafficking by capping and stabilizing actin filaments at plasmodesmata.

Here, we demonstrate that Arabidopsis thaliana Formin 2 (AtFH2) localizes to plasmodesmata (PD) through its transmembrane domain and is required for normal intercellular trafficking. Although loss-of-function atfh2 mutants have no overt developmental defect, PD's permeability and sensitivity to virus infection are increased in atfh2 plants. Interestingly, AtFH2 functions in a partially redundant manner with its closest homolog AtFH1, which also contains a PD localization signal. Strikingly, targeting of Class I formins to PD was also confirmed in rice, suggesting that the involvement of Class I formins in regulating actin dynamics at PD may be evolutionarily conserved in plants. In vitro biochemical analysis showed that AtFH2 fails to nucleate actin assembly but caps and stabilizes actin filaments. We also demonstrate that the interaction between AtFH2 and actin filaments is crucial for its function in vivo. These data allow us to propose that AtFH2 regulates PD's permeability by anchoring actin filaments to PD.

Sarcomeric and nonmuscle α-actinin isoforms exhibit differential dynamics at skeletal muscle Z-lines.

The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.

Filamins in Mechanosensing and Signaling

Filamins are essential, evolutionarily conserved, modular, multidomain, actin-binding proteins that organize the actin cytoskeleton and maintain extracellular matrix connections by anchoring actin filaments to transmembrane receptors. By cross-linking and anchoring actin filaments, filamins stabilize the plasma membrane, provide cellular cortical rigidity, and contribute to the mechanical stability of the plasma membrane and the cell cortex. In addition to binding actin, filamins interact with more than 90 other binding partners including intracellular signaling molecules, receptors, ion channels, transcription factors, and cytoskeletal and adhesion proteins. Thus, filamins scaffold a wide range of signaling pathways and are implicated in the regulation of a diverse array of cellular functions including motility, maintenance of cell shape, and differentiation. Here, we review emerging structural and functional evidence that filamins are mechanosensors and/or mechanotransducers playing essential roles in helping cells detect and respond to physical forces in their local environment.

Time-dependent Changes in Smooth Muscle Cell Stiffness and Focal Adhesion Area in Response to Cyclic Equibiaxial Stretch

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology.

The force is with α‐actinin: dynamic regulation of the extracellular matrix‐cytoskeletal connection in airway smooth muscle

The force is with α‐actinin: dynamic regulation of the extracellular matrix‐cytoskeletal connection in airway smooth muscle

Focusing on the Glomerular Slit Diaphragm: Podocin Enters the Picture

The nature of the glomerular filtration barrier is currently one of the most intensely studied and exciting problems in nephrology research, primarily because of the relatively recent discoveries that two novel and two previously known genes are intimately involved in glomerular filtration. The two novel genes, NPHS1 and NPHS2 (encoding nephrin and podocin, respectively) were identified by positional cloning. NPHS1 is mutated in congenital nephrotic syndrome of the Finnish type, 1 and NPHS2 is mutated in steroid resistant nephrotic syndrome. 2 The other two genes, previously characterized but not known to be involved in filtration, are ACTN4, encoding α-actinin-4, and Cd2ap, encoding CD2-associated protein (CD2AP). ACTN4 was found to be mutated in several families exhibiting inherited focal segmental glomerulosclerosis, 3 and knockout mice lacking CD2AP exhibit congenital nephrotic syndrome. 4 Several recent reviews have addressed the potential roles of these molecules in glomerular filtration. 5-10

Filopodial initiation and a novel filament-organizing center, the focal ring.

This study examines filopodial initiation and implicates a putative actin filament organizer, the focal ring. Filopodia were optically recorded as they emerged from veils, the active lamellar extensions of growth cones. Motile histories revealed three events that consistently preceded filopodial emergence: an influx of cytoplasm into adjacent filopodia, a focal increase in phase density at veil margins, and protrusion of nubs that transform into filopodia. The cytoplasmic influx probably supplies materials needed for initiation. In correlated time lapse-immunocytochemistry, these focal phase densities corresponded to adhesions. These adhesions persisted at filopodial bases, regardless of subsequent movements. In correlated time lapse-electron microscopy, these adhesion sites contained a focal ring (an oblate, donut-shaped structure approximately 120 nm in diameter) with radiating actin filaments. Filament geometry may explain filopodial emergence at 30 degree angles relative to adjacent filopodia. A model is proposed in which focal rings play a vital role in initiating and stabilizing filopodia: 1) they anchor actin filaments at adhesions, thereby facilitating tension development and filopodial emergence; 2) "axial" filaments connect focal rings to nub tips, thereby organizing filament bundling and ensuring the bundle intersects an adhesion; and 3) "lateral" filaments interconnect focal rings and filament bundles, thereby helping stabilize lamellar margins and filopodia.

The focal adhesions of chick retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells obtained from the eyes of chick embryos from colonies in vitro in which cells at the periphery of the colony express an undifferentiated, well-spread morphology and develop extremely large areas of cell-substratum adhesion. These adhesions can be classified as focal on the basis of the following: (a) their black surface reflection interference image, the contrast of which is not affected by changes in either the wavelength of the incident light or the refractive index of the immersion medium; (b) their association with the termini of actin-containing microfilament bundles; and (c) their ability to be labelled with antiserum against vinculin, a protein specific for adhesions of the focal type. The focal adhesions of RPE cells comprise laterally associated individual focal contacts, the mechanism by which this association is achieved and maintained is yet unknown. Because of the unusually large size and excellent microscopical definition of their focal adhesions, I used RPE cells to investigate the role of other actin-associated proteins in adhesion complexes. One of these, nonerythroid spectrin (fodrin), a protein suggested to play a role in anchoring actin filaments to the plasma membrane, was neither concentrated in nor excluded from the focal adhesions of RPE cells. Thus, at least in this cell type, spectrin seems unlikely to serve as a link between the major actin-containing microfilament bundles and the plasma membrane in the regions of cell-to-substratum contacts.

Platelet cytoskeleton alpha-actinin in normal and thrombasthenic platelets: distribution and immunologic characterization.

The subcellular localization of alpha-actinin (Mr 100,000) in human skeletal muscle is restricted to the Z line, in which it is believed to anchor actin filaments. Recently, this protein was identified in normal and thrombasthenic human platelets by its antigenic cross-reaction with antibodies to chicken gizzard alpha-actinin. In our study, the biochemical interaction between purified platelet alpha-actinin and striated muscle F-actin was examined by electron microscopy of negatively stained preparations. Like its muscle counterpart, platelet alpha-actinin promotes the cross-linking and bundling of actin filaments. Antibodies prepared to human platelet alpha-actinin cross-reacted with chicken gizzard alpha-actinin as shown by immunoelectrophoresis and the western blotting technique. Immunoblots prepared with normal and thrombasthenic platelets with antibodies to human platelet alpha-actinin revealed that this protein is susceptible to proteolysis. Extracts of freshly drawn platelets showed a protein band of 100 K. When the platelet extracts were incubated at 37 degrees C for various times, the immunoblots showed protein bands of 100 and 80 K. The proportion of the 80 K protein band increased with incubation time. This proteolysis can be prevented by chelating agents such as EDTA or the protease inhibitor leupeptin. Indirect immunofluorescent studies of human skin fibroblasts with antibodies to chicken gizzard actin and human skeletal muscle, chicken gizzard, and platelet alpha-actinin revealed the staining pattern characteristic of each protein. The distribution of alpha-actinin in normal and thrombasthenic platelets was assessed by ferritin-labeled immunoelectron microscopy. Ferritin particles were found in the cytoplasm immediately below the membrane and in some granules. There was no labeling associated with the mitochondria.

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KCl, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KCl and 2 mM MgCl 2 but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.

Alpha-Actinin and membrane glycoprotein IIIa are different proteins in human blood platelets.

It has been suggested that a platelet protein that is very similar to muscle alpha-actinin is identical to the membrane glycoprotein IIIa (GPIIIa) of platelets and is responsible for anchoring actin filaments directly into the plasma membrane of platelets. To determine if alpha-actinin and GPIIIa are related in platelets, we analyzed the purified proteins on 5% sodium dodecyl sulfate/polyacrylamide gels. The two proteins differ in mobility in both the unreduced and reduced states, and they stain differently with silver stain. In addition, alpha-actinin is a prominent component of the detergent-insoluble cytoskeletons of platelets, whereas GPIIIa is absent from these structures. By using monospecific antisera to the individual proteins, it was demonstrated that alpha-actinin and GPIIIa are immunologically distinct. We conclude that alpha-actinin and GPIIIa are different proteins in human blood platelets and that it is unlikely that alpha-actinin is an integral membrane protein.

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane.

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin-reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.