LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro‐inflammatory mediators. We examined mechanisms of LPS‐stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage‐like J774 cells. A wound‐healing assay revealed that LPS also activated directed cell movement that was followed by TNF‐α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5‐bisphosphate. Fractionation of Triton X‐100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin‐regulatory proteins, paxillin on tyrosine 118 by 80% and N‐WASP on serine 484/485 by 20%, and these events preceded activation of NF‐κB. LPS‐induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N‐WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS‐stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF‐α production suggesting that LPS‐induced cell motility is not required for TNF‐α release. J. Cell. Biochem. 113: 80–92, 2012. © 2011 Wiley Periodicals, Inc.     

Regulatory proteins alter nucleotide binding to acto-myosin of sliding filaments in motility assays

The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.     

Functions of LIM proteins in cell polarity and chemotactic motility

LimC and LimD are two novel LIM proteins of Dictyostelium, which are comprised of double and single LIM domains, respectively. Green fluorescent protein-fused LimC and LimD proteins preferentially accumulate at areas of the cell cortex where they co-localize with actin and associate transiently with cytoskeleton-dependent dynamic structures like phagosomes, macropinosomes and pseudopods. Furthermore, both LimC and LimD interact directly with F-actin in vitro. Mutant cells that lack either LimC or LimD, or both, exhibit normal growth. They are, however, significantly impaired in growth under stress conditions and are highly sensitive to osmotic shock, suggesting that LimC and LimD contribute towards the maintenance of cortical strength. Moreover, we noted an altered morphology and F-actin distribution in LimD(-) and LimC(-)/D(-) mutants, and changes in chemotactic motility associated with an increased pseudopod formation. Our results reveal both unique and overlapping roles for LimC and LimD, and suggest that both act directly on the actin cytoskeleton and provide rigidity to the cortex.     

Developmentally restricted actin-regulatory molecules control morphogenetic cell movements in the zebrafish gastrula

Although our understanding of the regulation of cellular actin and its control during the development of invertebrates is increasing, the question as to how such actin dynamics are regulated differentially across the vertebrate embryo to effect its relatively complex morphogenetic cell movements remains poorly understood. Intercellular signaling that provides spatial and temporal cues to modulate the subcellular localization and activity of actin regulatory molecules represents one important mechanism. Here we explore whether the localized gene expression of specific actin regulatory molecules represents another developmental mechanism. We have identified a cap1 homolog and a novel guanine nucleotide exchange factor (GEF), quattro (quo), that share a restricted gene expression domain in the anterior mesendoderm of the zebrafish gastrula. Each gene is required for specific cellular behaviors during the anterior migration of this tissue; furthermore, cap1 regulates cortical actin distribution specifically in these cells. Finally, although cap1 and quo are autonomously required for the normal behaviors of these cells, they are also nonautonomously required for convergence and extension movements of posterior tissues. Our results provide direct evidence for the deployment of developmentally restricted actin-regulatory molecules in the control of morphogenetic cell movements during vertebrate development.     

Regulatory GTP-binding proteins: emerging concepts on their role in cell function

The last few years have evidenced a tremendous expansion in our appreciation of the role of regulatory GTP-binding proteins in cellular activation. The availability of cholera and pertussis toxins to detect G proteins as well as methodological advances in the study of cellular function has afforded the opportunity to examine G protein participation in many cellular events. Regulation of adenylyl cyclase and cyclic GMP phosphodiesterase by G proteins has been demonstrated. Phosphatidylinositol-4,5-biphosphate specific phospholipase C activity appears to be subject to G protein control. G proteins regulate inward K+ and Ca2+ channels through a mechanism which may be independent of effects on the above mentioned enzymes. Certainly, the number of G proteins which have been identified from sequencing of complementary DNA affords the potential for G protein involvement in many cellular events. Only three G proteins have however been isolated and functionally characterized, Gs, Gi and transducin. Whether all the functions of these proteins have been identified remains to be seen.     

Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid

Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins (including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.     

Pure thoughts with impure proteins: Permeabilized cell models of organelle motility

Permeabilized cell models provide an experimental middle ground wherein the in vitro properties of mechanochemical proteins can be reconciled with the physical and topological constraints of the intact cell. Several well-studied examples of organelle motility are described here, including the actin-based cytoplasmic streaming of Characean algae, the microtubule-based aggregation and dispersion of pigment granules in chromatophores and the saltatory movements of vesicles along microtubules in fibroblasts and macrophages. The permeabilized models developed for these systems have helped to integrate observations in vivo with in vitro assays of motor proteins.     

Photo-induced conformational motility of proteins

The dynamics of proteins, detected by fluorescence, consists of three components: spontaneous dynamics, dipole-dipole photo-induced dynamics, thermal photo-induced dynamics. The photo-induced dynamics can lead to activation as well as inactivation of enzymes.     

Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter

Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.     

Directional motility of kinesin motor proteins

Kinesin motor proteins are molecules capable of moving along microtubules. They share homology in the so-called core motor domain which acts as a microtubule-dependent ATPase. The surprising finding that different members of the superfamily move in opposite directions along microtubules despite their close similarity has stimulated intensive research on the determinants of motor directionality. This article reviews recent biophysical, biochemical, structural and mutagenic studies that contributed to the elucidation of the mechanisms that cause directional motion of kinesin motor proteins.     

Regulatory proteins of vertebrate eye tissues

In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues.     

Regulatory mechanisms for ras proteins.

The proteins encoded by the ras proto-oncogenes play critical roles in normal cellular growth, differentiation and development in addition to their potential for malignant transformation. Several proteins that are involved in the control of the activity of p21ras have now been characterised. p120GAP stimulates the GTPase activity of p21ras and hence acts as a negative regulator of ras proteins. It may be controlled by tyrosine phosphorylation or association with tyrosine phosphorylated proteins. The neurofibromatosis type 1 (NF 1) gene also encodes a potential GTPase activating protein which is likely to be subject to a different control mechanism. Guanosine nucleotide exchange factors for p21ras have now been identified: these may be positive regulators of ras protein function. It appears that p21ras is subject to rapid regulation by several distinct mechanisms which are likely to vary in different cell types; the ras proteins are thereby able to act as very sensitive cellular monitors of the extracellular environment.     

Malaria IMC1 membrane skeleton proteins operate autonomously and participate in motility independently of cell shape

Plasmodium IMC1 (inner membrane complex 1) proteins comprise components of the subpellicular network, a lattice of intermediate filaments that form a structural part of the pellicle in the zoite stages of malaria parasites. Family members IMC1a and IMC1b are differentially expressed in sporozoites and ookinetes, respectively, but have functionally equivalent roles affecting cell morphology, strength, motility, and infectivity. Because of the coincident effects of previous imc1 gene disruptions on both zoite shape and locomotion, it has been impossible to ascribe a direct involvement in motility to these proteins. We show here that a third family member, IMC1h, has a distinct differential expression pattern and localizes to the pellicle of both ookinetes and sporozoites. Knock-out of IMC1h mimics the loss-of-function phenotypes of IMC1a and IMC1b in their respective life stages, indicating that IMC1 proteins could be operating co-dependently. By generating double null mutant parasites for IMC1h and IMC1b, we tested this hypothesis: double knock-out exacerbated the phenotypes of the single knock-outs in terms of ookinete strength, motility, and infectivity but did not further affect ookinete morphology. These findings provide the first genetic evidence that IMC1 proteins can function independently of each other and contribute to gliding motility independently of cell shape.