Cytoskeletal Protein Α-actinin Research Articles

Mechanical Activation of the β2-Adrenergic Receptor by Meningococcus: A Historical and Future Perspective Analysis of How a Bacterial Probe Can Reveal Signalling Pathways in Endothelial Cells, and a Unique Mode of Receptor Activation Involving Its N-Terminal Glycan Chains.

More than 12 years have passed since the seminal observation that meningococcus, a pathogen causing epidemic meningitis in humans, occasionally associated with infectious vasculitis and septic shock, can promote the translocation of β-arrestins to the cell surface beneath bacterial colonies. The cellular receptor used by the pathogen to induce signalling in host cells and allowing it to open endothelial cell junctions and reach meninges was unknown. The involvement of β-arrestins, which are scaffolding proteins regulating G protein coupled receptor signalling and function, incited us to specifically investigate this class of receptors. In this perspective article we will summarize the events leading to the discovery that the β2-adrenergic receptor is the receptor that initiates the signalling cascades induced by meningococcus in host cells. This receptor, however, cannot mediate cell infection on its own. It needs to be pre-associated with an “early” adhesion receptor, CD147, within a hetero-oligomeric complex, stabilized by the cytoskeletal protein α-actinin 4. It then required several years to understand how the pathogen actually activates the signalling receptor. Once bound to the N-terminal glycans of the β2-adrenergic receptor, meningococcus provides a mechanical stimulation that induces the biased activation of β-arrestin-mediated signalling pathways. This activating mechanical stimulus can be reproduced in the absence of any pathogen by applying equivalent forces on receptor glycans. Mechanical activation of the β2-adrenergic receptor might have a physiological role in signalling events promoted in the context of cell-to-cell interaction.

α-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions.

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.

Expression patterns of sarcomeric α-actin, α-actinin and UCP2 in the myocardium of Kunming mice after exposure to c-terminal polypeptide of cardiotrophin-1.

Cardiotrophin-1 (CT-1) activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expression pattern of sarcomeric contractile protein α-actin, specialized cytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 (UCP2) in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide (CP) of CT-1 (CT-1-CP, 500 μg·kg(-1)· day(-1)) for 1, 2, 3 and 4 week (s), respectively (4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group). Those injected with physiological saline for 4 weeks served as controls (n=10, with 5 males and 5 females). The heart tissues of mice were harvested at 1, 2, 3 or 4 week (s). Immunohistochemistry (IHC) and Western blotting (WB) were used to detect the distribution and expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyocytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The expression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group (IHC: χ(2)=6.125; WB: F=0.249, P>0.05) and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups (χ (2)=7.386, P>0.05). But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group (F=2.912; q=4.203, P<0.05). Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group (ICH: χ (2)=21.977; WB: F=50.388; P<0.01). The expression of UCP2 was initially increased (WB: control group vs. 1- or 2-week group, q values: 5.603 and 9.995, respectively, P<0.01) and then decreased (WB: control group vs. 3-week group, q=4.742, P<0.01; control group vs. 4-week group, q=0.558, P>0.05). It was suggested that long-term exposure to CT-1-CP could lead to the alteration in the expression of sarcomeric α-actin, α-actinin and mitochondrial UCP2. The different expressions of sarcomeric structure proteins and mitochondrial UCP2 may be involved in myocardial remodeling.

Sepsis-Induced Potentiation of Peritoneal Macrophage Migration Is Mitigated by Programmed Cell Death Receptor-1 Gene Deficiency

The effect of programmed cell death receptor-1 (PD-1) on phagocyte function has not been extensively described. Here we report that experimental mouse sepsis, cecal ligation and puncture (CLP), induced a marked increase in peritoneal macrophage random migration, motility and cell spread, but these changes were lost in the absence of PD-1. Alternatively, phagocytic activity was inversely affected. In vitro cell culture imaging studies, with the macrophage cell line J774, documented that blocking PD-1 with antibody led to aggregation of the cytoskeletal proteins α-actinin and F-actin. Further experiments looking at ex vivo peritoneal macrophages from mice illustrated that a similar pattern of α-actinin and F-actin was evident on cells from wild-type CLP mice but not PD-1-/- CLP mouse cells. We also observed that fMLP-induced migration by J774 cells was markedly attenuated using PD-1 blocking antibodies, a nonselective phosphatase inhibitor and a selective Ras-related protein 1 inhibitor. Finally, peritoneal macrophages derived from CLP as opposed to Sham mice demonstrated aspects of both cell surface co-localization with CD11b and internalization of PD-1 within vacuoles independent of CD11b staining. Together, we believe the data support a role for PD-1 in mediating aspects of innate macrophage immune dysfunction during sepsis, heretofore unappreciated.

Function of the syndecan-4 cytoplasmic domain in oligomerization and association with α-actinin in turkey muscle satellite cells

Syndecan-4 (S4) is a cell membrane heparan sulfate proteoglycan that plays a role in satellite cell mediated myogenesis. S4 modulates the proliferation of myogenic satellite cells, but the mechanism of how S4 functions during myogenesis is not well understood. In other cell systems, S4 has been shown to form oligomers in the cell membrane and interact through its cytoplasmic domain with the cytoskeletal protein α-actinin. This study addressed if S4 forms oligomers and interacts with α-actinin in muscle. The S4 cytoplasmic domain was found to interact with α-actinin in a phosphatidylinositol-4,5-bisphosphate dependent manner, but did not associate with vinculin. Through confocal microscopy, both S4 and syndecan-4 without the cytoplasmic domain were localized to the cell membrane. Although the cytoplasmic domain was necessary for the interaction with α-actinin, S4 oligomer formation occurred in the absence of the cytoplasmic domain. These data indicated that S4 function in skeletal muscle is mediated through the formation of oligomers and interaction with the cytoskeletal protein α-actinin.

Matrix metalloproteinase-2 degrades the cytoskeletal protein α-actinin in peroxynitrite mediated myocardial injury

Matrix metalloproteinase (MMP)-2 mediates myocardial ischemia–reperfusion injury which is characterized by enhanced peroxynitrite biosynthesis during early reperfusion. Direct infusion of peroxynitrite into isolated hearts activates MMP-2 prior to the loss in mechanical function. The mechanical dysfunction is prevented by MMPs inhibitors. MMP-2 is also found in the sarcomere of cardiomyocytes where it cleaves troponin I and myosin light chain I. Cytoskeletal proteins such as α-actinin, desmin and spectrin are found in close association with the sarcomere and are known to be degraded in ischemia–reperfusion injury. It remains unknown whether these proteins are degraded in peroxynitrite-induced myocardial injury and if cytoskeletal proteins are also targets for MMP-2. Peroxynitrite (80 μM) was infused into isolated rat hearts which led to a delayed onset but rapidly developing decline in mechanical function. The MMPs inhibitor PD-166793 or the peroxynitrite scavenger glutathione prevented the decline in cardiac function. At the end of perfusion, α-actinin levels were decreased by 45 ± 3% in peroxynitrite-infused hearts as compared to control hearts; however, this was normalized to that of control hearts with either PD-166793 or glutathione. Cardiac desmin and αII spectrin levels were unaltered following peroxynitrite infusion. α-Actinin and to a lesser extent desmin are susceptible to in vitro proteolysis by MMP-2 whereas spectrin is resistant. Cardiac dysfunction induced by peroxynitrite involves degradation of α-actinin that may be mediated by the proteolytic action of MMP-2.

The Cytoskeletal Proteins α-actinin, Ezrin, and Talin are De-expressed in Endometriosis and Endometrioid Carcinoma Compared With Normal Uterine Epithelium

In this retrospective study on banked tissue, we found that alpha-actinin and talin were completely de-expressed in both endometriosis and endometrioid carcinoma tissue. Some patchy, depolarized labeling for ezrin was noted in the endometrioid carcinoma but not in endometriosis. The loss of these proteins in both endometriosis and endometrioid carcinoma tissue indicates a significant change in the integrity of these tissues compared with normal and the possibility that individual cells may break away from the parent histology due to loss of cell adhesion. It also indicates a similarity between endometrioid cancer and endometriosis with respect to epithelial cell function and adhesion.

Phosphatidylinositol-4,5-Bisphosphate Regulates NMDA Receptor Activity through α-Actinin

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.

Phosphorylated α-Actinin and Protein-tyrosine Phosphatase 1B Coregulate the Disassembly of the Focal Adhesion Kinase·Src Complex and Promote Cell Migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.

Cytoplasmic anchorage of L-selectin controls leukocyte capture and rolling by increasing the mechanical stability of the selectin tether.

L-selectin is a leukocyte lectin that mediates leukocyte capture and rolling in the vasculature. The cytoplasmic domain of L-selectin has been shown to regulate leukocyte rolling. In this study, the regulatory mechanisms by which this domain controls L-selectin adhesiveness were investigated. We report that an L-selectin mutant generated by truncation of the COOH-terminal 11 residues of L-selectin tail, which impairs association with the cytoskeletal protein α-actinin, could capture leukocytes to glycoprotein L-selectin ligands under physiological shear flow. However, the conversion of initial tethers into rolling was impaired by this partial tail truncation, and was completely abolished by a further four-residue truncation of the L-selectin tail. Physical anchorage of both cell-free tail-truncated mutants within a substrate fully rescued their adhesive deficiencies. Microkinetic analysis of full-length and truncated L-selectin–mediated rolling at millisecond temporal resolution suggests that the lifetime of unstressed L-selectin tethers is unaffected by cytoplasmic tail truncation. However, cytoskeletal anchorage of L-selectin stabilizes the selectin tether by reducing the sensitivity of its dissociation rate to increasing shear forces. Low force sensitivity (reactive compliance) of tether lifetime is crucial for selectins to mediate leukocyte rolling under physiological shear stresses. This is the first demonstration that reduced reactive compliance of L-selectin tethers is regulated by cytoskeletal anchorage, in addition to intrinsic mechanical properties of the selectin–carbohydrate bond.

ENH, Containing PDZ and LIM Domains, Heart/Skeletal Muscle-Specific Protein, Associates with Cytoskeletal Proteins through the PDZ Domain

The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C β (PKCβ) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029–31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein α-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and α-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1–3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCβ is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCβ to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

Differential Protein Localization in Sarcomeric and Nonsarcomeric Contractile Structures of Cultured Cardiomyocytes

The use of cardiomyocyte cell culture models allows the identification of various cell mediators that bring about changes in subcellular structures and gene expression associated with hypertrophy. The effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), and triiodothyronine (T3) on gene expression and on the structural organization of myofibrillar and cytoskeletal proteins were compared in adult atrial (aARC) and ventricular (vARC) as well as in neonatal ventricular rat cardiomyocytes (vNRC) in long-term culture. Structural changes were evaluated by confocal microscopy and correlated to biochemical alterations. In vARC, IGF-I enhanced myofibrillar growth, whereas bFGF or T3 restricted sarcomere assembly to the central cell area, forming a sharp boundary in more than 50% of the cells. However, myosin occurred both in the cross-striated myofibrillar structures and in patches running along the nonsarcomeric fibrillar structures (also called stress fiber-like structures) in the cell periphery. In cells treated with either bFGF or T3, the expression of α-smooth muscle actin (α-sm actin) was greatly increased. This actin isoform was incorporated mainly into the nonsarcomeric contractile structures outside the area where myofibrils ended abruptly. α-sm actin protein increased up to 14- to 17-fold while the mRNA showed a moderate increase of 2- to 4-fold. This suggests that α-sm actin is mainly regulated at the translational or posttranslational level. In contrast, the cytoskeletal proteins α-actinin and vinculin increased only moderately (less than 2-fold) but also showed a relocalization in cells with restricted myofibrils. In aARC and in vNRC, α-sm actin was only moderately upregulated by bFGF or T3 and no drastic morphological changes were observed. In conclusion, IGF-I, bFGF, and T3 induced characteristic structural phenotypes depending on the type of cardiomyocyte. Large amounts of α-sm actin as expressed in bFGF and T3 treated vARC seem to be incompatible with sarcomere assembly.

Activation of SRC family kinases in human neutrophils. Evidence that p58 c-frg and p53/56 lyn redistributed to a Triton X-100-insoluble cytoskeletal fraction, also enriched in the caveolar protein Caveolin, display an enhanced kinase activity

Protein tyrosine phosphorylation is one of the signals involved in stimulation of neutrophil (PMN) functions. We found that phorbol myristate acetate (PMA) activates the src family tyrosine kinases p58 c−fgr and p53/56 lyn in suspended PMNs. Moreover, we found that up to about 20% of p58 c−fgr and p53/56 lyn redistribute to a Triton X-100-insoluble fraction after PMA stimulation, and it is this fraction of the two kinases which displays an increased activity. These changes of p58 c−fgr and p53/56 lyn distribution and activity correlated with tyrosine phosphorylation of endogenous substrates. In fact, in PMA-stimulated PMNs tyrosine phosphorylated proteins are mostly recovered in a Triton-insoluble cell fraction. To separate cytoskeletal from caveolar structures, which both display Triton X-100-insolubility, we used the detergent n-octyl ß- d-glucopyranoside (OGP) which solubilises components of caveolae. We found that the caveolae marker protein, caveolin, as well as the cytoskeletal protein α-actinin and p58 c−fgr and p53/56 lyn, is insoluble in OGP. These findings suggest that PMA stimulation promotes the formation of multimolecular complexes containing cytoskeletal proteins, caveolin-containing structures and src family protein tyrosine kinases. Moreover, they show that p58 c−fgr and p53/56 lyn associated with this multimolecular complex display an enhanced kinase activity.

Identification of the cytoskeletal protein α-actinin as a platelet thrombospondin-binding protein

Binding of the α-granular thrombospondin (TSP) to the plasma membrane of activated platelets has long been documented, yet the molecular mechanism involved in its secretion and surface expression have not been elucidated. Using a ligand blot binding assay where electrophoretically separated platelet proteins were incubated with purified 125I-labeled TSP, we observed a strong interaction of [ 125I]TSP with a 100 kDa single chain protein. On performing a platelet subfractionation, the 100 kDa protein was predominantly localized in the cytosol from which it was purified by preparative electrophoresis and was identified by amino acid sequencing to the cytoskeletal protein, α-actinin. We further demonstrated that [ 125I]TSP interacts with α-actinin in a specific manner and with a high affinity ( K d = 6.6nM) in a solid-phase binding assay.