Characterisation of Encephalitozoon cuniculi α-actinin.

We have recently described marked differences in cell migration rates and organization of actin in human melanoma cell lines isolated from various stages of tumor progression. Metastatic lines derived from lymph node metastases organized actin into stress fiber arrays and had high mean migration rates in vitro when compared to lines from other stages. Melanoma cells also reveal marked differences in localization of alpha-actinin and beta 1 integrins at stress fiber termination sites (focal contacts). Disruption of this organization is induced by antibodies against beta 1 integrins, alpha-actinin, recently postulated as having a role in linkage of actin to beta 1 integrins, is differentially expressed in melanoma cells by Northern blot analysis and a relatively high alpha-actinin to actin ratio is associated with stress fiber formation and increased cell migration. Furthermore, actin-binding protein, which cross-links actin filaments, is also significantly increased in lines exhibiting high migration rates. Control of migration and actin organization may be mediated by extracellular matrices and/or modulation of actin-associated proteins including alpha-actinin and actin binding protein. These findings provide evidence that an interaction of transmembrane adhesion molecules and elements of the cytoskeleton in melanoma cells may be responsible for differences in migration rates and capacity for metastasis.

Erythrocyte tropomodulin (E-Tmod or Tmod1) of 41 kDa is a tropomyosin (TM)-binding protein that caps the slow-growing end of the actin filaments. Its N-terminal half is flexible, whereas the C-terminal half has a single domain structure. E-Tmod/TM5 complex may function as a "molecular ruler" generating actin protofilaments of ∼37 nm. Here we report the discovery of a short isoform of 29 kDa that lacks the N-terminal actin-binding domain (N-ABD) but retains the C-terminal actin-binding domain (C-ABD). E-Tmod29 can be generated by alternative splicing from an upstream promoter or by multiple transcriptional start sites from a downstream promoter. Promoter switching leads to a surge of E-Tmod41 in reticulocytes, which degrades quickly in the cytosol. We expressed recombinant isoforms in Escherichia coli and tested their binding toward TM5, G-actin, and F-actin. Solid-phase binding assays show that, without the N-terminal 102 residues, E-Tmod29 binds to TM5 or G-actin more strongly than E-Tmod41 does, but barely binds to F-actin after TM5 binding. Differential bindings explain the distinct localizations of E-Tmod29 in the cytosol and E-Tmod41 on the membrane. Sequential bindings and immunofluorescent staining further suggest that 1) TM5 binding to E-Tmod41 may open up the flexible N-terminal half, exposing N-ABD and unblocking C-ABD; 2) N-ABD binds to F-actin and C-ABD binds to G-actin; and 3) F-actin binding to N-ABD may prevent G-actin from binding to C-ABD. E-Tmod29 may thus modulate the availability of TM5 and G-actin for E-Tmod41 to construct the protofilament-based membrane skeletal network for circulating erythrocytes.

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and alpha-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. alpha-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo.

Molecular Structure of the Rod Domain of Dictyostelium Filamin

This study describes the effect of actin-binding protein derived from rabbit lung macrophages on the mechanical properties of F-actin. The dynamic storage modulus, G'(omega), and loss modulus, G"(omega) of F-actin, at concentrations from 1 to 4 mg/ml, in the absence or presence of actin-binding protein at molar ratios to actin of 1:1000 to 1:125, were measured at frequencies ranging from 3 X 10(-3) to 0.5 Hz. Actin-binding protein increased the dynamic moduli of F-actin, but this increase was much greater as either the actin-binding protein/actin ratio or the total protein concentration increased. Moreover, there was a convergence of the values of G' and G" at high frequencies for F-actin which became more prominent upon the addition of actin-binding protein. The value of the modulus obtained by an extrapolation of these data to actin concentrations similar to that found in the cell cortex was close to values which have been obtained by direct measurements. The addition of actin-binding protein to an F-actin solution enabled it to reach an equilibrium strain following the application of a stress, in contrast to pure F-actin. These data allow a more rigorous definition of the "sol" to "gel" transition and suggest that the cross-linking of actin filaments by actin-binding protein leads to the formation of a network structure whose underlying mechanism of mechanical behavior is short range intrafilament bending in contrast to the classical rubber network.

Many actin-binding proteins have been purified from different cell types, and a number of them share certain features: they are affected by calcium in their interaction with actin and have similar subunit molecular weights (∼100,000) on SDS-poly-acrylamide gels. These proteins have been given various names, including gelsolin1–3, villin4,5, actinogelin6,7, Dictyostelium 95K protein8,9, platelet 95K protein10 and Acanthamoeba 85K protein11. The actin-binding protein α-actinin has received little attention in this analysis, even though it has a subunit molecular weight in this range (∼100,000) and has been shown immunologically to exist in non-muscle cells12,13. Here we report that α-actinin purified from HeLa cells is inhibited from cross-linking actin filaments by micromolar free calcium. In many properties it closely resembles actinogelin, Dictyostelium 95K protein and Acanthamoeba 85K protein, but it appears distinct from proteins such as gelsolin and villin which, in the presence of calcium, fragment actin filaments into short oligomers3,5. We conclude that in this molecular weight range there are at least two classes of non-muscle actin-binding protein: one corresponding to non-muscle α-actinins and the other to proteins such as gelsolin and villin.

Many actin-binding proteins are expressed in eukaryotic cells. These polypeptides assist in stabilizing and rearranging the organization of the actin cytoskeleton in response to external stimuli, or during cell migration and adhesion. Here we review a particular set of actin-binding proteins called plastins. Plastins (also called fimbrins) belong to a subclass of actin-binding proteins known as actin bundling proteins. Three isoforms have been characterized in mammals: T-plastin is expressed in cells from solid tissue, whereas L-plastin occurs predominantly in hematopoietic cells. The third isoform, I-plastin, is specifically expressed in the small intestine, colon and kidney. These proteins share the unique property of cross-linking actin filaments into tight bundles. Although plastins are primarily involved in regulation of the actin cytoskeleton, they possess some unique features. For instance, they are implicated in invasion by pathogenic bacteria such as Shigella flexneri and Salmonella typhimurium. Also, L-plastin plays an important role in leukocyte function. T-plastin, on the other hand, is possibly involved in DNA repair. Finally, both T- and L-plastin are implicated in several diseases, and L-plastin is considered to be a valuable marker for cancer.

The structure of the N-terminal actin-binding domain of human dystrophin and how mutations in this domain may cause Duchenne or Becker muscular dystrophy

Fimbrins/plastins have been implicated in the generation of distinct actin structures, which are linked to different cellular processes. Historically, fimbrins/plastins were mainly considered as generating tight actin bundles. Here, we demonstrate that different members of the fimbrin/plastin family have diverged biochemically during evolution to generate either tight actin bundles or loose networks with distinct biochemical and biophysical properties. Using the phylogenetically and functionally distinct Arabidopsis fimbrins FIM4 and FIM5 we found that FIM4 generates both actin bundles and cross-linked actin filaments, whereas FIM5 only generates actin bundles. The distinct functions of FIM4 and FIM5 are clearly observed at single-filament resolution. Domain swapping experiments showed that cooperation between the conformationally plastic calponin-homology domain 2 (CH2) and the N-terminal headpiece determines the function of the full-length protein. Our study suggests that the structural plasticity of fimbrins/plastins has biologically meaningful consequences, and provides novel insights into the structure-function relationship of fimbrins/plastins as well as shedding light on how cells generate distinct actin structures.

Filamin A (FLNa) cross-links actin filaments (F-actin) into three-dimensional gels in cells, attaches F-actin to membrane proteins, and is a scaffold that collects numerous and diverse proteins. We report that Ca(2+)-calmodulin binds the actin-binding domain (ABD) of FLNa and dissociates FLNa from F-actin, thereby dissolving FLNa.F-actin gels. The FLNa ABD has two calponin homology domains (CH1 and CH2) separated by a linker. Recombinant CH1 but neither FLNa nor its ABD binds Ca(2+)-calmodulin in the absence of F-actin. Extending recombinant CH1 to include the negatively charged region linker domain makes it, like full-length FLNa, unable to bind Ca(2+)-calmodulin. Ca(2+)-calmodulin does, however, dissociate the FLNa ABD from F-actin provided that the CH2 domain is present. These findings identify the first evidence for direct regulation of FLNa, implicating a mechanism whereby Ca(2+)-calmodulin selectively targets the FLNa.F-actin complex.

α-Actinin displays homology with erythroid and non-erythroid spectrins, dystrophin, and utrophin. These proteins all share a similar domain structure, with an N-terminal actin-binding domain, a variable number of triple α-helical spectrin-like repeats, and a pair of C-terminal EF hands. The actin-binding domain of α-actinin is homologous to that in filamin, fimbrin, and ABP 120, and there is a more limited match with the N-terminal domain of calponin.

Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM). However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD). We created and expressed the wild-type (WT) N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts), and unfolds faster than the WT (stopped-flow kinetics). Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments). These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

The linkage of heterodimeric (alpha/beta) integrin receptors with their extracellular matrix ligands and intracellular actin cytoskeleton is a fundamental step for controlling cell adhesion and migration. Binding of the actin-linking protein, talin, to integrin beta cytoplasmic tails (CTs) induces high affinity ligand binding (integrin activation), whereas binding of another actin-linking protein, filamin, to the integrin beta CTs negatively regulates this process by blocking the talin-integrin interaction. Here we show structurally that migfilin, a novel cytoskeletal adaptor highly enriched in the integrin adhesion sites, strongly interacts with the same region in filamin where integrin beta CTs bind. We further demonstrate that the migfilin interaction dissociates filamin from integrin and promotes the talin/integrin binding and integrin activation. Migfilin thus acts as a molecular switch to disconnect filamin from integrin for regulating integrin activation and dynamics of extracellular matrix-actin linkage.

Proteomic analysis of ACTN4-interacting proteins reveals it’s a putative involvement in mRNA metabolism

Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain