Actin-binding Protein Α-actinin Research Articles

Abstract P1058: Whole-cell Mechanical Loading And Unloading Triggers More Post-translational Modifications In α-actinin Than Myosin Activators And Inhibitors

The hypothesis tested is that unloading of mechanical forces affects acetylation, ubiquitination, and phosphorylation of the actin-binding protein α-actinin located in the Z-disc. We applied targeted proteomics to interrogate the post-translational modification changes during loading and unloading. Cell morphology and post-translational modifications were determined in a mechanical intervention consisting of 1 Hz cyclic strain for 24 hr (loaded) followed by 6 hr rest (unloaded) of cultured neonatal rat ventricular myocytes (NRVMs). This was compared to a chemical intervention consisting of treating NRVMs with the myosin inhibitor Mavacamten (1 μM, 6hr) or activator Omecamtiv Mecarbil (0.5 μM, 6hr). Quantitative immunofluorescence showed both chemical and mechanical loading increased α-actinin content while Mavacamten decreased the α-actinin content. Mass spectrometry analysis of affinity-purified α-actinin revealed that the mechanical intervention led to increased levels of post-translational modifications compared with the chemical interventions. Specifically, α-actinin ubiquitination increased with mechanical loading-unloading; acetylation decreased with mechanical loading-unloading and increased with mechanical loading; and phosphorylation remained unchanged with mechanical loading-unloading but increased with mechanical loading. Fluorescence recovery after photobleaching (FRAP) experiments demonstrated that Mavacamten increased the dynamics of overexpressed YFP-tagged α-actinin and a GFP-tagged CapZ in NRVMs when compared to Omecamtiv Mecarbil treatments and controls. Overall, the results suggest a link between sarcomere homeostasis and mechanical forces via mechanisms involving acetylation, phosphorylation and ubiquitination of α-actinin and a second Z-disc protein, CapZ. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH grants HL151825 (CS) and HL62426 (RJS, BR, and CMW).

Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia.

Successful pregnancy requires normal placentation, which largely depends on the tight regulation of proliferation, invasion, and migration of trophoblast cells. Abnormal functioning of trophoblast cells may cause failure of uterine spiral artery remodeling, which may be related to pregnancy-related disorders, such as preeclampsia. Here, we reported that an actin-binding protein, α-actinin (ACTN)4, was dysregulated in placentas from early onset preeclampsia. Moreover, knockdown of ACTN4 markedly inhibited trophoblast cell proliferation by reducing AKT membrane translocation. Furthermore, E-cadherin regulated ACTN4 and β-catenin colocalization on trophoblast cell podosomes, and ACTN4 down-regulation suppressed the E-cadherin-induced cell invasion increase via depolymerizing actin filaments. Moreover, loss of ACTN4 recapitulated a number of the features of human preeclampsia. Therefore, our data indicate that ACNT4 plays a role in trophoblast function and is required for normal placental development.-Peng, W., Tong, C., Li, L., Huang, C., Ran, Y., Chen, X., Bai, Y., Liu, Y., Zhao, J., Tan, B., Luo, X., Wang, H., Wen, L., Zhang, C., Zhang, H., Ding, Y., Qi, H., Baker, P. N. Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia.

Tyrosine Phosphorylation of CD13 Regulates Inflammatory Cell–Cell Adhesion and Monocyte Trafficking

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Competition between α-actinin and Ca2+-Calmodulin Controls Surface Retention of the L-type Ca2+ Channel CaV1.2

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²⁺ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²⁺ influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²⁺-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²⁺ influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²⁺-calmodulin triggers Ca²⁺-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²⁺ influx.

EWI-2 Association with α-Actinin Regulates T Cell Immune Synapses and HIV Viral Infection

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.

Colocalization of -actinin and Synaptopodin in the Pyramidal Cell Axon Initial Segment

The cisternal organelle that resides in the axon initial segment (AIS) of neocortical and hippocampal pyramidal cells is thought to be involved in regulating the Ca(2+) available to maintain AIS scaffolding proteins, thereby preserving normal AIS structure and function. Through immunocytochemistry and correlative light and electron microscopy, we show here that the actin-binding protein α-actinin is present in the typical cistenal organelle of rodent pyramidal neurons as well as in a large structure in the AIS of a subpopulation of layer V pyramidal cells that we have called the "giant saccular organelle." Indeed, this localization of α-actinin in the AIS is dependent on the integrity of the actin cytoskeleton. Moreover, in the cisternal organelle of cultured hippocampal neurons, α-actinin colocalizes extensively with synaptopodin, a protein that interacts with both actin and α-actinin, and they appear concomitantly during the development of these neurons. Together, these results indicate that α-actinin and the actin cytoskeleton are important components of the cisternal organelle that are probably required to stabilize the AIS.

The microtubule inhibiting agent epothilone B antagonizes glioma cell motility associated with reorganization of the actin-binding protein α-actinin 4

Invasion of normal brain tissue by brain tumor cells is a major contributing factor to the recurrence and resistance of clinically diagnosed glioblastomas to therapy (surgery, chemotherapy, radiation). Here, we have assessed the efficacy of the microtubule inhibiting agent epothilone B on glioblastoma cell motility, a prerequisite cellular program of invasive glioblastomas. Using cell migration assays and immunofluorescence techniques we demonstrated that epothilone B abrogated glioblastoma cell motility as a consequence of α-actinin 4 redistristrubiton and the breakdown of cellular structures (leading edge, stress fibers) it is associated with during cell migration. Evaluation of the microtubule actin cross linking factor in glioblastoma cells also revealed epothilone B invoked changes in this cytoskeleton cross linking protein, resembling α-actinin 4 changes in response to epothilone B. We have demonstrated in this study that epothilone B antagonizes glioblastoma cell motility due to the disruption of cytoskeleton binding proteins that aide in preserving the structural organization of the cytoskeleton filamentous network. Furthermore, we provide preclincial evidence that epothilone B effects on glioblastomas are not limited to the impairment of dividing tumors cells but that it also targets migratory and invasive glioblastoma cells, suggesting that this agent has potential clinical benefit due to its ability to target divergent cellular programs in the glioblastoma tumor mass.

α-Actinin Is Required for the Proper Assembly of Z-Disk/Focal-Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans

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.

Actin-binding Protein α-Actinin-1 Interacts with the Metabotropic Glutamate Receptor Type 5b and Modulates the Cell Surface Expression and Function of the Receptor

Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

Amino Acid Residues within EnterohemorrhagicEscherichia coliO157:H7 Tir Involved in Phosphorylation, α-Actinin Recruitment, and Nck-Independent Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) adherence to epithelial cells results in the formation of actin pedestals. Pedestal formation requires the bacterial protein Tir, which is inserted into the epithelial cell plasma membrane by the type III secretion system. EPEC and EHEC use different Tir-based mechanisms for pedestal formation, and the EPEC Tir residues required have been well described. In contrast, little is known about the regions of EHEC O157:H7 Tir that are essential for pedestal formation. Additionally, EHEC O157:H7 Tir is serine/threonine phosphorylated, although the residues involved and their role in pedestal formation are not known. In this study, we describe two regions within the carboxy terminus of EHEC O157:H7 Tir that are required for phosphorylation and pedestal formation. Serines 436 and 437 are substrates for protein kinase A phosphorylation, although this is not required to form pedestals. Using a series of internal deletion mutants, we found that amino acids 454 to 463 are required for efficient pedestal formation. Deleting this region resulted in a significant decrease in the recruitment of both filamentous actin and the actin binding protein alpha-actinin. As alpha-actinin binds directly to the EHEC O157:H7 amino terminus, these data suggest that its recruitment is dependent on pedestal formation.

The Adenosine A2A Receptor Interacts with the Actin-binding Protein α-Actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.

Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.

Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.

Non-muscle alpha actinins are calcium-sensitive actin-binding proteins.

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.