Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.     

Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development

During metamorphic development, bullfrogs (Rana catesbeiana) undergo substantial morphological, anatomical, and physiological changes as the animals prepare for the transition from a fully-aquatic to a semi-terrestrial existence. Using BrdU incorporation and immunohistochemistry, we quantify changes in cell proliferation in two key auditory brainstem nuclei, the dorsolateral nucleus and the superior olivary nucleus, over the course of larval and early postmetamorphic development. From hatchling through early larval stages, numbers of proliferating cells increase in both nuclei, paralleling the overall increase in total numbers of cells available for labeling. Numbers of proliferating cells in the superior olivary nucleus decrease during the late larval and deaf periods, and significantly increase during metamorphic climax. Proliferating cells in the dorsolateral nucleus increase in number from hatchling to late larval stages, decrease during the deaf period, and increase during climax. In both nuclei, numbers of proliferating cells decrease during the postmetamorphic froglet stage, despite increases in the number of cells available for label. Newly generated cells express either glial- or neural-specific phenotypes beginning between 1 week and 1 month post-BrdU injection, respectively, while some new cells express gamma-aminobutyric acid within 2 days of mitosis. Our data show that these auditory nuclei dramatically up-regulate mitosis immediately prior to establishment of a transduction system based on atmospheric hearing.     

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self-renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg-induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg-induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.     

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Background

Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.

Methodology/Principal Findings

We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.

Conclusions/Significance

Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.     

New Horned Dinosaurs from Utah Provide Evidence for Intracontinental Dinosaur Endemism

Background

During much of the Late Cretaceous, a shallow, epeiric sea divided North America into eastern and western landmasses. The western landmass, known as Laramidia, although diminutive in size, witnessed a major evolutionary radiation of dinosaurs. Other than hadrosaurs (duck-billed dinosaurs), the most common dinosaurs were ceratopsids (large-bodied horned dinosaurs), currently known only from Laramidia and Asia. Remarkably, previous studies have postulated the occurrence of latitudinally arrayed dinosaur “provinces,” or “biomes,” on Laramidia. Yet this hypothesis has been challenged on multiple fronts and has remained poorly tested.

Methodology/Principal Findings

Here we describe two new, co-occurring ceratopsids from the Upper Cretaceous Kaiparowits Formation of Utah that provide the strongest support to date for the dinosaur provincialism hypothesis. Both pertain to the clade of ceratopsids known as Chasmosaurinae, dramatically increasing representation of this group from the southern portion of the Western Interior Basin of North America. Utahceratops gettyi gen. et sp. nov.—characterized by short, rounded, laterally projecting supraorbital horncores and an elongate frill with a deep median embayment—is recovered as the sister taxon to Pentaceratops sternbergii from the late Campanian of New Mexico. Kosmoceratops richardsoni gen. et sp. nov.—characterized by elongate, laterally projecting supraorbital horncores and a short, broad frill adorned with ten well developed hooks—has the most ornate skull of any known dinosaur and is closely allied to Chasmosaurus irvinensis from the late Campanian of Alberta.

Conclusions/Significance

Considered in unison, the phylogenetic, stratigraphic, and biogeographic evidence documents distinct, co-occurring chasmosaurine taxa north and south on the diminutive landmass of Laramidia. The famous Triceratops and all other, more nested chasmosaurines are postulated as descendants of forms previously restricted to the southern portion of Laramidia. Results further suggest the presence of latitudinally arrayed evolutionary centers of endemism within chasmosaurine ceratopsids during the late Campanian, the first documented occurrence of intracontinental endemism within dinosaurs.     

Structural Optimization and De Novo Design of Dengue Virus Entry Inhibitory Peptides

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding “pseudoenergies”, we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 µM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery.     

The Structure and Function of Bacterial Actin Homologs

During the past decade, the appreciation and understanding of how bacterial cells can be organized in both space and time have been revolutionized by the identification and characterization of multiple bacterial homologs of the eukaryotic actin cytoskeleton. Some of these bacterial actins, such as the plasmid-borne ParM protein, have highly specialized functions, whereas other bacterial actins, such as the chromosomally encoded MreB protein, have been implicated in a wide array of cellular activities. In this review we cover our current understanding of the structure, assembly, function, and regulation of bacterial actins. We focus on ParM as a well-understood reductionist model and on MreB as a central organizer of multiple aspects of bacterial cell biology. We also discuss the outstanding puzzles in the field and possible directions where this fast-developing area may progress in the future.The discovery of cytoskeletal proteins in bacteria has fundamentally altered our understanding of the organization and evolution of bacteria as cells. Homologs of eukaryotic actin represent the most molecularly and functionally diverse family of bacterial cytoskeletal elements. Recent phylogenetic studies have identified more than 20 subgroups of bacterial actin homologs (Derman et al. 2009) (Fig. 1). Many of these bacterial actins are encoded on extrachromosomal plasmids, but most bacterial species with nonspherical morphologies also encode chromosomal actin homologs (Daniel and Errington 2003). The two earliest proteins to be characterized as bacterial actins were the chromosomal protein MreB (Jones et al. 2001) and the plasmidic protein ParM (Jensen and Gerdes 1997). MreB and ParM remain the best-characterized of the bacterial actins and we will thus focus on these two proteins for most of this article.Open in a separate windowFigure 1.The superfamily of bacterial actin homologs. Shown is a phylogenetic tree of the bacterial actin subfamilies that have been identified to date based on sequence homology. The subfamilies that have been experimentally shown to polymerize are labeled and colored. (Courtesy of Joe Pogliano, based on Derman et al. 2009.)The appreciation that bacteria possess actin homologs only occurred in the past decade. MreB was first identified as a protein involved in cell shape regulation in Escherichia coli in the late 1980s (Doi et al. 1988). In the early 1990s, pioneering bioinformatic studies identified similarities in a group of ATPases that have five conserved motifs (Bork et al. 1992), a feature dubbed the actin superfamily fold. Although this group includes actin and MreB, it also contains proteins that do not polymerize into filaments, such as sugar kinases like hexokinase and chaperones like Hsp70. A number of bacterial proteins are present in the actin superfamily, including the bacterial cell division protein FtsA which interacts with the tubulin homolog FtsZ and may or may not form filaments in different contexts (van den Ent and Lowe 2000). Because MreB did not appear significantly more related to actin than these nonfilamentous proteins, the weak sequence similarity with actin was largely ignored for the better part of a decade. This changed in 2001 when two seminal papers showed that Bacillus subtilis MreB forms cytoskeletal filaments in vivo (Jones et al. 2001) and that Thermotoga maritima MreB forms cytoskeletal filaments in vitro (van den Ent et al. 2001). Indeed, structural and biochemical studies of both MreB and ParM have convincingly showed that these proteins closely resemble actin and polymerize into linear filaments in a nucleotide-dependent manner (Fig. 2).Open in a separate windowFigure 2.Structures of F-actin (Holmes et al. 1990), MreB (van den Ent et al. 2001), and ParM (van den Ent et al. 2002). (Left) Structures of F-actin filaments (PDB entry 1YAG). (Second from the left) MreB filaments from T. maritima (PDB entry 1JCE). (Center) ParM:ADP monomer in the “closed” conformation. (Second from the right) apo ParM monomer in the “open” conformation. (Right) ParM filament. Shown are the position of the nucleotide within the interdomain cleft, the conservation of fold, and the axis of the protofilament extension (arrow). Note that the conformational change shown for ParM from the “open” to “closed” state is predicted for all actin homologs. (Adapted, with permission from, Michie and Löwe 2006.)Research following the identification of bacterial cytoskeletal proteins has focused on understanding their assembly, regulation, and function. Here, we will summarize our current understanding of these issues and highlight the outstanding questions. We will begin with ParM, whose well-characterized assembly and dynamics represent a model for future studies of all cytoskeletal proteins. We will then focus on MreB, whose diverse activities appear to be central to the cell biology of many bacterial species.     

Purification of reversibly oxidized proteins (PROP) reveals a redox switch controlling p38 MAP kinase activity

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.