Isolation and properties of brain alpha-actinin.
Ontology highlight
ABSTRACT:
SUBMITTER: Schook W
PROVIDER: S-EPMC1186041 | biostudies-other | 1978 Oct
REPOSITORIES: biostudies-other
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Project description:The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin.
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Project description:The mechanisms by which living cells respond to mechanical stimuli are not yet fully understood. It has been suggested that mechanosensing proteins play an important role in mechanotransduction because their binding affinities are directly affected by the external stress. α-Actinin is an actin cross-linker and may act as a mechanosensor in adhesion sites. Its interaction with vinculin is suggested to be mechanically regulated. In this study, the free energy of activation is explored using the umbrella sampling method. An activation trajectory is generated in which α-actinin's vinculin-binding site swings out of the rod domain, leading to approximately an 8 kcal/mol free energy release. The activation trajectory reveals several local and global conformational changes along the activation pathway accompanied by the breakage of a number of key interactions stabilizing the inhibited structure. These results may shed light on the role of α-actinin in cellular mechanotransduction and focal adhesion formation.
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Project description:Due to alternative splicing, the human ACTN1 gene codes for three different transcripts of α-actinin; one isoform that is expressed only in the brain and two with a more general expression pattern. The sequence difference is located to the C-terminal domains and the EF-hand motifs. Therefore, any functional or structural distinction should involve this part of the protein. To investigate this further, the calcium affinities of these three isoforms of α-actinin 1 have been determined by isothermal calorimetry.
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Project description:Alpha-actinin and vinculin orchestrate reorganization of the actin cytoskeleton following the formation of adhesion junctions. alpha-Actinin interacts with vinculin through the binding of an alpha-helix (alphaVBS) present within the R4 spectrin repeat of its central rod domain to vinculin's N-terminal seven-helical bundle domain (Vh1). The Vh1:alphaVBS structure suggests that alphaVBS first unravels from its buried location in the triple-helical R4 repeat to allow it to bind to vinculin. alphaVBS binding then induces novel conformational changes in the N-terminal helical bundle of Vh1, which disrupt its intramolecular association with vinculin's tail domain and which differ from the alterations in Vh1 provoked by the binding of talin. Surprisingly, alphaVBS binds to Vh1 in an inverted orientation compared to the binding of talin's VBSs to vinculin. Importantly, the binding of alphaVBS and talin's VBSs to vinculin's Vh1 domain appear to also trigger distinct conformational changes in full-length vinculin, opening up distant regions that are buried in the inactive molecule. The data suggest a model where vinculin's Vh1 domain acts as a molecular switch that undergoes distinct structural changes provoked by talin and alpha-actinin binding in focal adhesions versus adherens junctions, respectively.
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Project description:Despite the central role PSD-95 plays in anchoring postsynaptic AMPARs, how PSD-95 itself is tethered to postsynaptic sites is not well understood. Here we show that the F-actin binding protein α-actinin binds to the very N terminus of PSD-95. Knockdown (KD) of α-actinin phenocopies KD of PSD-95. Mutating lysine at position 10 or lysine at position 11 of PSD-95 to glutamate, or glutamate at position 53 or glutamate and aspartate at positions 213 and 217 of α-actinin, respectively, to lysine impairs, in parallel, PSD-95 binding to α-actinin and postsynaptic localization of PSD-95 and AMPARs. These experiments identify α-actinin as a critical PSD-95 anchor tethering the AMPAR-PSD-95 complex to postsynaptic sites.
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Project description: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.
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Project description:BackgroundAnnexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state.ResultsTo identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST)- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α)-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP) tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by α-actinin immunostaining in shRNA treated cells.ConclusionsIn overall, the present study demonstrated for the first time that annexin A6 physically interacts with sarcomeric α-actinin and alters contractility of cardiomyocytes suggesting that it might play important role in excitation and contraction process.
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Project description:The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.
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Project description:Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.
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