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On the edge: modeling protrusion.

Actin-based protrusion is the first step in cell crawling. In the last two decades, the studies of actin networks in the lamellipodium and Listeria's comet tail advanced so far that the last goal of the reductionist agenda - reconstitution of protrusion from purified components in vitro and in silico - became viable. Earlier models dealt with growth of and force generation by a single actin filament. Modern models of tethered ratchet, autocatalytic branching, end-tracking motor action and elastic- and nano- propulsion have recently helped to elucidate dynamics and forces in complex actin networks. By considering these models, their limitations and their relationships to recent biophysical data, progress is being made toward a unified model of protrusion.

Actin Cytoskeleton↗

Protein complexes regulating Arp2/3-mediated actin assembly.

Key steps in regulating actin dynamics are the de novo nucleation and elongation of actin filaments, which can be catalysed by a limited number of proteins and protein complexes. Among these, Arp2/3 complex and formins are the best studied. Arp2/3-complex activity is controlled through signalling-dependent association with nucleation-promoting factors, such as the WASP/WAVE family proteins. A common theme for these molecules, which is well established for WAVEs but is only just beginning to emerge for WASPs, is that they act as coincident detectors of a variety of signalling pathways through the formation of large multi-molecular complexes.

Actin-Related Protein 2-3 Complex↗

Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components.

Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.

Actin-Related Protein 2-3 Complex↗

Pathogens: bacterial needles ruled to length and specificity.

The mechanisms behind length regulation of prokaryotic surface structures has long eluded microbiologists. The recent identification of a protein that functions as a 'molecular ruler' to determine the physical length of a bacterial extracellular needle advances our understanding of surface structure biogenesis.

Bacterial Proteins↗

Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins.

Planar polarity development in the Drosophila wing is under the control of the frizzled (fz) pathway. Recent work has established that the planar polarity (PP) proteins become localized to either the distal, proximal, or both sides of wing cells. Fz and Dsh distal accumulation is thought to locally activate the cytoskeleton to form a hair . Planar polarity effector (PPE) genes such as inturned (in) are not required for the asymmetric accumulation of PP proteins, but they are required for this to influence hair polarity. in mutations result in abnormal hair polarity and are epistatic to mutations in the PP genes. We report that In localizes to the proximal side of wing cells in a PP-dependent and PP-instructive manner. We further show that the function of two other PPE genes (fuzzy and fritz) is essential for In protein localization, a finding consistent with previous genetic data that suggested these three genes function in a common process. These data indicate that accumulation of proteins at the proximal side of wing cells is a key event for the distal activation of the cytoskeleton to form a hair.

Animals↗

Podosome and sealing zone: specificity of the osteoclast model.

The bone resorption function of osteoclasts is dependent on the integrity of the actin cytoskeleton. Depending on the substratum upon which the osteoclasts are spread, there are two different structures of actin known as podosomes and the sealing zone. To understand the specific properties and relationship of podosomes and the sealing zone, we used live-cell imaging of cultured osteoclasts. When cultured on extracellular matrix components, podosomes in these cells are organized in higher-ordered structures. These are clustered podosomes that will arrange later into dynamic short-lived rings which finally expand to the cell periphery to form a stable long-lived podosome belt in fully differentiated cells. In osteoclasts, this specific podosome patterning is under the control of microtubules (MTs). Indeed, nocodazole treatment does not affect podosome formation but only the transition between clusters/rings and belts. During this transition, MTs accumulate a specific post-translational modification of tubulin by acetylation. This process is repressed by an inhibitory pathway involving the GTPase Rho, its effector mDIA2 and the recently discovered tubulin deacetylase HDAC6. The specific function of this acetylation is still unknown but is also observed in active osteoclasts forming a sealing zone which is also MT dependent. Thus, it appears that the podosome belt is reminiscent of the sealing zone. Indeed, podosome belts and sealing zones are characterized by their overall stability. Despite their similar behavior, a sealing zone is not formed by fusion of podosomes. The formation of a podosome belt or a sealing zone is controlled by the external environment. Indeed, only the bone mineral fraction, known as apatite crystal, is able to induce sealing zone formation in mature osteoclasts. Contact of osteoclasts with apatite stimulates the non-receptor tyrosine kinase c-Src and the GTPase Rho in order to form the sealing zone. As we will discuss in this review, it appears that podosomes and the sealing zone are strikingly linked.

Actin Cytoskeleton↗

Invadopodia and podosomes in tumor invasion.

Cell migration through the extracellular matrix (ECM) is necessary for cancer cells to invade adjacent tissues and metastasize to an organ distant from primary tumors. Highly invasive carcinoma cells form ECM-degrading membrane protrusions called invadopodia. Tumor-associated macrophages have been shown to promote the migratory phenotypes of carcinoma cells, and macrophages are known to form podosomes, similar structures to invadopodia. However, the role of invadopodia and podosomes in vivo remains to be determined. In this paper, we propose a model for possible functions and interactions of invadopodia and podosomes in tumor invasion, based on observations that macrophage podosomes degrade ECM and that podosome formation is regulated by colony-stimulating factor-1 signaling.

Actin Cytoskeleton↗

Convergent evolution within the genus Solanum: the specialised anther cone develops through alternative pathways.

Many angiosperm species produce cones of anthers which release pollen through pores in response to vibration by pollinating bees ("buzz-pollination"). Anther cones of varying degrees of strength are a defining morphological trait for the genus Solanum. Anthers arranged in a robust ('pepper pot') cone are restricted to a single clade within the genus, and may therefore be assumed to be monophyletic. We show that in some species within this clade, such as tomato (Solanum lycopersicum), the anther cone is held together by interlocking hairs (trichomes) along the edges of the anthers. In other species within the clade, such as woody nightshade (Solanum dulcamara), the expanded anther surfaces are closely appressed to form the tightly bound cone, strengthened by extracellular secretions. Ectopic expression of the MIXTA gene from Antirrhinum majus in S. dulcamara results in the formation of ectopic trichomes on the anthers which cause the cone to disintegrate. Therefore, these two species produce the same macroscopic structure through two mutually exclusive developmental routes and the robust anther cone is derived differently within the clade. This example demonstrates that convergence between closely related species can be easily mistaken for homology, and may thus be underestimated.

Adaptation, Physiological↗

Structural analysis of Mycoplasma pneumoniae by cryo-electron tomography.

Bacteria of the genus Mycoplasma lack obvious homologs of prokaryotic or eukaryotic cytoskeletal, as well as motility-related genes (except FtsZ). Nevertheless, they maintain characteristic cell shapes and show adhesion and gliding abilities on both artificial surfaces and cells. Earlier genetic, biochemical, and electron microscopic analyses have shown that the tip structure, located at the tapered end of gliding mycoplasmas, is indispensable for this behavior. In this study, we have analyzed the fine structure of the Mycoplasma pneumoniae tip by cryo-electron tomography. We show that the central rod is surrounded by quasi-periodical electron-dense macromolecular complexes. Additional complexes are located at the distal end of the rod which connect the rod to the cytoplasmic membrane. Furthermore, we detect a structure at the proximal end of the rod that attaches the rod to the cell membrane. The surface protein complexes have been mapped in detail and their distribution on the cell surface has been visualized. Since the rod structures were detected at a close to native state of the cells, they allow us to build a hypothesis describing the motility mechanism of M. pneumoniae. Finally, we have evaluated the ribosome density of the organism by a template matching approach, whereby the reliability of the detection was supported by a comparative bioinformatics analysis.

Antigens, Surface↗

An avian influenza vaccine for humans targeting the polymerase B2 protein inside the capsid instead of hemagglutinin or neuramidase on the virus surface.

Vaccines for avian influenza typically are aimed at hemagglutinin or neuramidase on the outside of the virus capsid. A major problem with such an approach is that the genes coding for these proteins have a very rapid mutation rate, forcing commercial producers to wait for mutations to occur before developing effective new versions of standard vaccines. However, a recent study has revealed that the 1918 flu virus, like the H5N1 avian flu virus, has an E627K mutation in its polymerase B2 component, which is located inside the virus capsid. Other research has indicated that this mutation strongly influences the virulence of H5N1. It seems reasonable to believe that the constancy, over more than 80 years, of the E627K mutation could be exploited to begin developing a vaccine now, rather than waiting for new mutations. Consequently, a publicly available database at the National Center for Biotechnology Information (NCBI) website, and the SYFPEITHI online computer algorithm, were used to generate a hypothesis about a peptide-based vaccine targeted at the E627K mutation in PB2 of the avian influenza virus. It was found that the peptide sequence, DTVQIIKLL, present in the PB2 protein of the H5N1 virus, would be expected to bind to HLA-A26 restricted immune system cell surface receptors. Hence, the bound peptide might be capable of stimulating protection from cytotoxic T lymphocytes. Should the present hypothesis be confirmed in laboratory studies, and an effective vaccine developed for individuals expressing the HLA-A26 receptor; further research would be indicated. This research would be aimed at determining whether molecular modifications to the DTVQIIKLL peptide could make it effective with other members of the HLA-A1 supertype to which HLA-A26 belongs. In addition to allowing vaccine development to begin now, this peptide-based approach would have the advantage of avoiding the use of dangerous, live, avian influenza virus during mass production.

Capsid Proteins↗

Aurigamonas solis n. gen., n. sp., a soil-dwelling predator with unusual helioflagellate organisation and belonging to a novel clade within the Cercozoa.

A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.

Animals↗

Role of bacterial cell surface structures in Escherichia coli biofilm formation.

Various cell surface molecules and structures have been implicated in biofilm formation in Escherichia coli. This review presents an overview of the occurrence, production and interaction of these components, their influence at one or more developmental stages of biofilm formation, and their potential role as virulence factors in pathogenic E. coli strains.

Biofilms↗

Carvedilol-mediated antioxidant protection against doxorubicin-induced cardiac mitochondrial toxicity.

The cardiotoxicity associated with doxorubicin (DOX) therapy limits the total cumulative dose and therapeutic success of active anticancer chemotherapy. Cardiac mitochondria are implicated as primary targets for DOX toxicity, which is believed to be mediated by the generation of highly reactive free radical species of oxygen from complex I of the mitochondrial electron transport chain. The objective of this study was to determine if the protection demonstrated by carvedilol (CV), a beta-adrenergic receptor antagonist with strong antioxidant properties, against DOX-induced mitochondrial-mediated cardiomyopathy [Toxicol. Appl. Pharmacol. 185 (2002) 218] is attributable to its antioxidant properties or its beta-adrenergic receptor antagonism. Our results confirm that DOX induces oxidative stress, mitochondrial dysfunction, and histopathological lesions in the cardiac tissue, all of which are inhibited by carvedilol. In contrast, atenolol (AT), a beta-adrenergic receptor antagonist lacking antioxidant properties, preserved phosphate energy charge but failed to protect against any of the indexes of DOX-induced oxidative mitochondrial toxicity. We therefore conclude that the cardioprotective effects of carvedilol against DOX-induced mitochondrial cardiotoxicity are due to its inherent antioxidant activity and not to its beta-adrenergic receptor antagonism.

Adrenergic beta-Antagonists↗

Movin' on up: the role of PtdIns(4,5)P(2) in cell migration.

Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.

Animals↗

MgcRacGAP regulates cortical activity through RhoA during cytokinesis.

Although Rho GTPases regulate multiple cellular events, their role in cell division is still obscure. Here we show that expression of a GTPase-activating protein (GAP)-deficient mutant (R386A) of the Rho regulator MgcRacGAP induces abnormal cortical activity during cytokinesis in U2OS cells. Multiple large blebs were observed in cells expressing MgcRacGAP R386A from the onset of anaphase to the late stage of cell division. When mitotic blebbing was excessive, cytokinesis was inhibited, and cells with micronuclei were generated. It has been reported that blebbing is caused by abnormal cortical activity. The MgcRacGAP R386A-induced abnormal cortical activity was inhibited by the dominant negative form of RhoA, but not Rac1 or Cdc42. Moreover, expression of constitutively active RhoA also induced drastic cortical activity during cytokinesis. Unlike apoptotic blebbing, MgcRacGAP R386A-induced blebbing was not inhibited by the ROCK inhibitor Y-27632, suggesting that MgcRacGAP regulates cortical activity during cytokinesis through a novel signaling pathway. We propose that MgcRacGAP plays a pivotal role in cytokinesis by regulating cortical movement through RhoA.

Cell Division↗

Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells.

As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.

Animals↗

MEGAP impedes cell migration via regulating actin and microtubule dynamics and focal complex formation.

Over the past several years, it has become clear that the Rho family of GTPases plays an important role in various aspects of neuronal development including cytoskeleton dynamics and cell adhesion processes. We have analysed the role of MEGAP, a GTPase-activating protein that acts towards Rac1 and Cdc42 in vitro and in vivo, with respect to its putative regulation of cytoskeleton dynamics and cell migration. To investigate the effects of MEGAP on these cellular processes, we have established an inducible cell culture model consisting of a stably transfected neuroblastoma SHSY-5Y cell line that endogenously expresses MEGAP albeit at low levels. We can show that the induced expression of MEGAP leads to the loss of filopodia and lamellipodia protrusions, whereas constitutively activated Rac1 and Cdc42 can rescue the formation of these structures. We have also established quantitative assays for evaluating actin dynamics and cellular migration. By time-lapse microscopy, we show that induced MEGAP expression reduces cell migration by 3.8-fold and protrusion formation by 9-fold. MEGAP expressing cells also showed impeded microtubule dynamics as demonstrated in the TC-7 3x-GFP epithelial kidney cells. In contrast to the wild type, overexpression of MEGAP harbouring an artificially introduced missense mutation R542I within the functionally important GAP domain did not exert a visible effect on actin and microtubule cytoskeleton remodelling. These data suggest that MEGAP negatively regulates cell migration by perturbing the actin and microtubule cytoskeleton and by hindering the formation of focal complexes.

Actins↗