Synemin interacts with the LIM domain protein zyxin and is essential for cell adhesion and migration

Synemin is a unique cytoplasmic intermediate filament protein for which there is limited understanding of its exact cellular functions. The single human synemin gene encodes at least two splice variants named α-synemin and β-synemin, with the larger α-synemin containing an additional 312 amino acid insert within the C-terminal tail domain. We report herein that, by using the entire tail domain of the smaller β-synemin as the bait in a yeast two-hybrid screen of a human skeletal muscle cDNA library, the LIM domain protein zyxin was identified as an interaction partner for human synemin. The synemin binding site in human zyxin was subsequently mapped to the C-terminal three tandem LIM-domain repeats, whereas the binding site for zyxin within β-synemin is within the C-terminal 332 amino acid region (SNβTII) at the end of the long tail domain. Transient expression of SNβTII within mammalian cells markedly reduced zyxin protein level, blocked localization of zyxin at focal adhesion sites and resulted in decreased cell adhesion and increased motility. Knockdown of synemin expression with siRNAs within mammalian cells resulted in significantly compromised cell adhesion and cell motility. Our results suggest that synemin participates in focal adhesion dynamics and is essential for cell adhesion and migration.     

Tandem zyxin LIM sequences do not enhance force sensitive accumulation

The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity.     

The extracellular domain of herpes simplex virus gE is sufficient for accumulation at cell junctions but not for cell-to-cell spread

Herpes simplex virus (HSV) expresses a number of membrane glycoproteins, including gB, gD, and gH/gL, that function in both entry of virus particles and movement of virus from an infected cell to an uninfected cell (cell-to-cell spread). However, a complex of HSV glycoproteins gE and gI (gE/gI) is required for efficient cell-to-cell spread, especially between cells that form extensive cell junctions, yet it is not necessary for entry of extracellular virions. We previously showed that gE/gI has the capacity to localize specifically to cell junctions; the glycoprotein complex was found at lateral surfaces of cells in contact with other cells but not at those lateral surfaces not forming junctions or at apical surfaces. By virtue of these properties, gE/gI is an important molecular handle on the poorly understood process of cell-to-cell spread. Here, we show that the cytoplasmic domain of gE is important for the proper delivery of gE/gI to lateral surfaces of cells. Without this domain, gE/gI is found on the apical surface of epithelial cells, and more uniformly in the cytoplasm, although incorporation into the virion envelope is unaffected. However, even without proper trafficking signals, a substantial fraction of gE/gI retained the capacity to accumulate at cell junctions. Therefore, the extracellular domain of gE can mediate accumulation of gE/gI at cell junctions, if the glycoprotein can be delivered there, probably through interactions with ligands on the opposing cell. The role of phosphorylation of the cytoplasmic domain of gE was also studied. A second mutant HSV type 1 was constructed in which three serine residues that form a casein kinase II phosphorylation site were changed to alanine residues, reducing phosphorylation by 70 to 80%. This mutation did not affect accumulation at cell junctions or cell-to-cell spread.     

Opposing roles of zyxin/LPP ACTA repeats and the LIM domain region in cell-cell adhesion

Cadherins mediate cell-cell adhesion by linking cell junctions to actin networks. Although several actin regulatory systems have been implicated in cell-cell adhesion, it remains unclear how such systems drive cadherin-actin network formation and how they are regulated to coincide with initiation of adhesion. Previous work implicated VASP in assembly of cell-cell junctions in keratinocytes and the VASP-binding protein zyxin colocalizes with VASP at cell-cell junctions. Here we examine how domains in zyxin and its relative LPP contribute to cell-cell junction assembly. Using a quantitative assay for cell-cell adhesion, we demonstrate that zyxin and LPP function to increase the rate of early cell-cell junction assembly through the VASP-binding ActA repeat region. We also identify the LIM region of zyxin and LPP to be a regulatory domain that blocks function of these proteins. Deletion of the LIM domains drives adhesion and increases VASP level in detergent insoluble cadherin-actin. Dominant-negative zyxin/LPP mutants reduce the rate of adhesion, lower VASP levels in detergent-insoluble cadherin-actin networks, and allow for the accumulation of capping protein at cell-cell contacts. These data implicate the LIM domains of zyxin and LPP in regulating cell-cell junction assembly through VASP.     

The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability

Cdc37 is a molecular chaperone required for folding of protein kinases. It functions in association with Hsp90, although little is known of its mechanism of action or where it fits into a folding pathway involving other Hsp90 cochaperones. Using a genetic approach with Saccharomyces cerevisiae, we show that CDC37 overexpression suppressed a defect in v-Src folding in yeast deleted for STI1, which recruits Hsp90 to misfolded clients. Expression of CDC37 truncation mutants that were deleted for the Hsp90-binding site stabilized v-Src and led to some folding in both sti1Delta and hsc82Delta strains. The protein kinase-binding domain of Cdc37 was sufficient for yeast cell viability and permitted efficient signaling through the yeast MAP kinase-signaling pathway. We propose a model in which Cdc37 can function independently of Hsp90, although its ability to do so is restricted by its normally low expression levels. This may be a form of regulation by which cells restrict access to Cdc37 until it has passed through a triage involving other chaperones such as Hsp70 and Hsp90.     

The LIM protein Ajuba influences p130Cas localization and Rac1 activity during cell migration

Cell migration requires extension of lamellipodia that are stabilized by formation of adhesive complexes at the leading edge. Both processes are regulated by signaling proteins recruited to nascent adhesive sites that lead to activation of Rho GTPases. The Ajuba/Zyxin family of LIM proteins are components of cellular adhesive complexes. We show that cells from Ajuba null mice are inhibited in their migration, without associated abnormality in adhesion to extracellular matrix proteins, cell spreading, or integrin activation. Lamellipodia production, or function, is defective and there is a selective reduction in the level and tyrosine phosphorylation of FAK, p130Cas, Crk, and Dock180 at nascent focal complexes. In response to migratory cues Rac activation is blunted in Ajuba null cells, as detected biochemically and by FRET analysis. Ajuba associates with the focal adhesion-targeting domain of p130Cas, and rescue experiments suggest that Ajuba acts upstream of p130Cas to localize p130Cas to nascent adhesive sites in migrating cells thereby leading to the activation of Rac.     

Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.     

An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment

The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for alpha-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21-42 specifically binds to alpha-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with alpha-actinin in blot overlays and fail to recruit alpha-actinin in living cells. When zyxin lacking the alpha-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the alpha-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.     

The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein

The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58 kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.     

The cytoplasmic domain of Ig alpha is necessary and sufficient to support efficient early B cell development

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.     

The extracellular domain of receptor activity-modifying protein 1 is sufficient for calcitonin receptor-like receptor function

A functional calcitonin gene-related peptide (CGRP) receptor requires dimerization of calcitonin receptor-like receptor (CRLR) with receptor activity-modifying protein 1 (RAMP 1). To determine the function of the three domains (extracellular, ECD; transmembrane, TM; and tail domains) of human RAMP 1, three mutants were constructed: RAMP 1 without the cytoplasmic tail, a chimera consisting of the ECD of RAMP 1 and the TM and tail of the platelet-derived growth factor receptor, and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for their ability to associate with CRLR to effect CGRP-stimulated cAMP accumulation, CGRP binding, CRLR trafficking, and cell surface expression. All RAMP 1 mutants were able to associate with CRLR with full efficacy for CGRP-stimulated cAMP accumulation. However, the RAMP 1/platelet-derived growth factor receptor chimera demonstrated a 10-fold decrease in potency for CGRP signaling and binding, and the RAMP 1-ECD mutant had a 4000-fold decrease in potency. In conclusion, the ECD of RAMP 1 is sufficient for normal CRLR association and efficacy. The presence of a TM domain and the specific sequence of the RAMP 1 TM domain contribute to CGRP affinity and potency. The C-terminal tail of RAMP 1 is unnecessary for CRLR function.     

The interactome of LIM domain proteins: the contributions of LIM domain proteins to heart failure and heart development

LIM domain proteins all contain at least one double zinc-finger motif. They belong to a large family and here we review those expressed mainly in mammalian hearts, but particularly in cardiomyocytes. These proteins contain between one and five LIM domains and usually these proteins contain other domains that have specific functions such as actin-binding, kinases and nuclear translocation motifs. While several recent reviews have summarised the importance of individual LIM domain proteins, this is the first review of its kind to cover all LIMs associated with the heart. Here we examine 33 LIM proteins (including three that bind to, but do not themselves contain, LIM domains) that are implicated in either the development of the heart, heart disorders and failure, or both. Our analysis is consistent with the view that cardiac LIM domain proteins form multiple extensive networks of multi-protein complexes within the myocardium. This multiplicity of binding partners probably protects the heart as it is challenged to maintain cardiac output, until the imbalance reaches a turning point that results in failure. We believe that the complexity of LIM interactions is properly described by the term LIM interactome.     

The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.     

Mouse cytomegalovirus M33 is necessary and sufficient in virus-induced vascular smooth muscle cell migration

Mouse cytomegalovirus (MCMV) encodes two potential seven-transmembrane-spanning proteins with homologies to cellular chemokine receptors, M33 and M78. While these virus-encoded chemokine receptors are necessary for the in vivo pathogenesis of MCMV, the function of these proteins is unknown. Since vascular smooth muscle cell (SMC) migration is of critical importance for the development of atherosclerosis and other vascular diseases, the ability of M33 to promote SMC motility was assessed. Similar to human CMV, MCMV induced the migration of mouse aortic SMCs but not mouse fibroblasts. To demonstrate whether M33 was required for MCMV-induced SMC migration, we employed interfering-RNA technology to specifically knock down M33 expression in the context of viral infection. The knockdown of M33 resulted in the specific reduction of M33 protein expression and ablation of MCMV-mediated SMC migration but failed to reduce viral growth in cultured cells. Adenovirus vector expression of M33 was sufficient to promote SMC migration, which was enhanced in the presence of recombinant mouse RANTES (mRANTES). In addition, M33 promoted the activation of Rac1 and extracellular signal-related kinase 1/2 upon stimulation with mRANTES. These findings demonstrate that mRANTES is a ligand for this chemokine receptor and that the activation of M33 occurs in a ligand-dependent manner. Thus, M33 is a functional homologue of US28 that is required for MCMV-induced vascular SMC migration.     

One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein

The N-terminal repeat (SAC1) of the S-protein of Lactobacillus acidophilus bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not. Treatment of CWFs with hydrofluoric acid, but not phenol, prevented binding. Apparently, SAC1 is necessary and sufficient for cell wall binding. Our data suggest that SAC anchors the S-protein to a cell wall teichoic acid.