Table of Contents

Volume 5, Issue 1

  January/February 2011

  Pages 1 - 95

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JOURNAL CLUB

Open Access Article

First evidence of RNA interference mechanism induced in human: Getting closer to a cure?

Lise Roth
Pages 1 - 3
http://dx.doi.org/10.4161/cam.5.1.13490
Abstract | PDF

Knocking down a human gene by RNA silencing has been the focus of a number of laboratories since the discovery of the RNAi mechanism more than 10 years ago. In their remarkable work, M. Davis and collaborators show for the first time the induction of an RNAi pathway in humans following systemic administration of nanoparticles. In a clinical trial, they demonstrate the accumulation of the particles in the tumor, as well as the downregulation of mRNA and protein expression of the target gene. Moreover, they successfully linked these observations to the induction of mRNA cleavage by the delivered siRNA in the tumor.

Open Access Article

Integrin α_IIbβ₃: A novel effector of Gα₁₃

Ramya Chari-Turaga and Ulhas P. Naik
Pages 4 - 5
http://dx.doi.org/10.4161/cam.5.1.13559
Abstract | PDF

Under physiological conditions, circulating platelets are discoid in shape. On these platelets, the fibrinogen receptor (integrin αIIbβ3) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin αIIbβ3 through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling. Outside-in signaling control processes such as platelet spreading and clot retraction by regulating small G-proteins such as RhoA, Rac and cdc42.




COMMENTARIES & VIEWS

Open Access Article

BCL2 interaction with actin in vitro may inhibit cell motility by enhancing actin polymerization

Hengning Ke, Jennifer Y. Zhang, Steven K. Akiyama and John E. French
Pages 6 - 10
http://dx.doi.org/10.4161/cam.5.1.13175
Abstract | PDF

In addition to its well-defined role as an antagonist in apoptosis, we propose that BCL2 may act as an intracellular suppressor of cell motility and adhesion under certain conditions. Our evidence shows that, when over-expressed in both cancer and non-cancer cells, BCL2 can form a complex with actin and gelsolin that functions to decrease gelsolin-severing activity to increase actin polymerization, and, thus, suppress cell adhesive processes. The linkage between increased BCL2 and increased actin polymerization on the one hand, and suppression of cell adhesion, spreading, and motility on the other hand, is a novel observation that may provide a plausible explanation for why BCL2 over-expression in some tumors is correlated with improved patient survival. In addition, we have identified conditions in vitro in which F-actin polymerization can be increased while cell motility is reduced. These findings underscore the possibility that BCL2 may be involved in modulating cytoskeleton reorganization, and may provide an opportunity to explore signal transduction pathways important for cell adhesion and migration and to develop small molecule therapies for suppression of cancer metastasis.

Open Access Article

RHO methylation matters: A role for isoprenylcysteine carboxylmethyltransferase in cell migration and adhesion

Ian Cushman and Patrick J. Casey
Pages 11 - 15
http://dx.doi.org/10.4161/cam.5.1.13196
Abstract | PDF

Numerous proteins involved in diverse aspects of cell biology undergo a process of post-translational modification termed prenylation. The prenylation pathway consists of three enzymatic steps, the final of which is methylation of the carboxyl-terminal prenylcysteine formed in the first two steps by the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt). Due to the prevalence of prenylated proteins in cancer biology, and the findings that several of the proteins are involved in processes controlling cell migration and adhesion, we sought to examine the role of Icmt - mediated methylation on cell behavior associated with metastasis. We found that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading were also impaired by Icmt inhibition. Further investigation revealed that inhibition of Icmt significantly decreased the activation of both RhoA and Rac1, which are both prenylated proteins. The data obtained were consistent with the decreased activation being due to an increase in Rho GDP-dissociation inhibitor (GDI) binding by both proteins in the absence of their methylation. Importantly, the addition of exogenous RhoA or Rac1 to cells in which Icmt was inhibited was able to partially, but selectively, rescue directed and random migration, respectively. These results establish a role for Icmt-mediated methylation in cell migration, and point to specific prenylated proteins involved in this biology. The prenylation pathway has been targeted for oncogenic therapies, but the role of methylation in cell motility had been largely unexplored until now. The finding that methylation of Rho family members impacts on a specific component of their function provides an additional avenue through which to interrogate the biology of this important class of regulatory proteins.

Open Access Article

Acto-myosin based response to stiffness and rigidity sensing

Jonathan Fouchard, Démosthène Mitrossilis and Atef Asnacios
Pages 16 - 19
http://dx.doi.org/10.4161/cam.5.1.13281
Abstract | PDF

Cells sense the rigidity of their environment and respond to it. Most studies have been focused on the role of adhesion complexes in rigidity sensing. In particular, it has been clearly shown that proteins of the adhesion complexes were stretch-sensitive, and could thus trigger mechano-chemical signaling in response to applied forces. In order to understand how this local mechano-sensitivity could be coordinated at the cell scale, we have recently carried out single cell traction force measurements on springs of varying stiffness. We found that contractility at the cell scale (force, speed of contraction, mechanical power) was indeed adapted to external stiffness, and reflected ATPase activity of non-muscle myosin II and acto-myosin response to load. Here we suggest a scenario of rigidity sensing where local adhesions sensitivity to force could be coordinated by adaptation of the acto-myosin dependent cortical tension at the global cell scale. Such a scenario could explain how spreading and migration are oriented by the rigidity of the cell environment.

Open Access Article

Adhesion molecules: Key players in mesenchymal stem cell-mediated immunosuppression

Guangwen Ren, Arthur I. Roberts and Yufang Shi
Pages 20 - 22
http://dx.doi.org/10.4161/cam.5.1.13491
Abstract | PDF

Adhesion molecules are known to be important components of an active T cell-mediated immune response. Signals generated at a site of inflammation cause circulating T-cells to respond by rolling, arrest, and then transmigration through the endothelium, all of which are mediated by adhesion molecules. Consequently, strategies have been developed to treat immune disorders with specific antibodies that block the interaction of adhesion molecules. However, the therapeutic effects of such remedies are not always achieved. Our recent investigations have revealed that intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) work together with chemokines to induce immunosuppression mediated by mesenchymal stem cells (MSCs), thus demonstrating the dual role of adhesion molecules in immune responses. Since MSCs represent an important component of the stromal cells in an inflammatory microenvironment, our findings provide novel information for understanding the regulation of immune responses and for designing new strategies to treat immune disorders.

Open Access Article

ZF21 is a new regulator of focal adhesion disassembly and a potential member of the spreading initiation center

Makoto Nagano, Daisuke Hoshino, Takeharu Sakamoto, Toshifumi Akizawa, Naohiko Koshikawa and Motoharu Seiki
Pages 23 - 28
http://dx.doi.org/10.4161/cam.5.1.13492
Abstract | PDF

Adherent cells migrate on extracellular matrices (ECM) by repeated spreading and contraction of the cell body. Focal adhesions (FAs) play a major role in the adherence of cells to the ECM and in the generation of the cellular forces that maintain morphology and allow cells to move. FAs also mediate bidirectional transmembrane signals in conjunction with growth factor receptors and signaling molecules. Although the mechanisms that regulate cell migration are not yet fully understood, the regulation of the formation and turnover of FAs is a key factor determining the rate and direction of cell migration. We recently identified a component of FAs termed ZF21, which is a member of a family of proteins characterized by the presence of a conserved phosphoinositide-binding motif. ZF21 promotes dephosphorylation of FAK at Tyr³⁹⁷ upon microtubule extension to FAs and thereby regulates the disassembly of FAs in a microtubules-dependent manner. To obtain further insight into the regulation of cell adhesion by ZF21, we analyzed proteins associating with ZF21 by proteomic analysis. We identified 45 proteins including FA-related proteins and multiple RNA binding proteins that have been shown recently to be components of the spreading initiation center (SIC). SICs are cell adherent structures that can be observed only in the early stages of cell spreading and have been implicated in regulating the rate of cell spreading. In this article, we report new ZF21-binding proteins identified by proteomic analysis and discuss the potential functions of ZF21 in regulating disassembly of FAs.

Open Access Article

Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles

Ricardo Bentes Azevedo, Caroline Rodrigues Alves Valois, Sacha Braun Chaves, Jaqueline Rodrigues Silva and Monica Pereira Garcia
Pages 29 - 33
http://dx.doi.org/10.4161/cam.5.1.13558
Abstract | PDF

Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA up-regulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen, and increased concentration of tumor necrosis factor-α in lung, in a time dependent manner. The critical relevance of the β2 integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identifies β2 integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.

Open Access Article

The botulinum toxin complex meets E-cadherin on the way to its destination

Yo Sugawara and Yukako Fujinaga
Pages 34 - 36
http://dx.doi.org/10.4161/cam.5.1.13574
Abstract | PDF

Botulinum neurotoxin (BoNT) causes the disease botulism, which is characterized by flaccid paralysis, in humans and animals. The metalloprotease activity of BoNT inhibits neurotransmitter release at neuro-muscular junctions. In most cases, poisoning occurs when BoNT is ingested. Therefore, BoNT must pass through the epithelial barrier of the gastrointestinal tract to enter the systemic circulation and reach the target site. BoNT forms large protein complexes by associating with non-toxic components referred to as non-toxic non-hemagglutinin (NTNH) and hemagglutinin (HA). These proteins protect BoNT from the low pH and proteases in the digestive tract. We recently determined that HA has an unexpected function of disrupting the intercellular epithelial barrier by directly binding to E-cadherin. HA binds to E-cadherin and disrupts its function in a species-specific manner, and this interaction is essential to disrupt tight junctions. This activity is thought to facilitate the absorption of BoNT through the paracellular route of the intestinal epithelium in susceptible species.

Open Access Article

Modulators of endothelial cell filopodia: PECAM-1 joins the club

Horace M. DeLisser
Pages 37 - 41
http://dx.doi.org/10.4161/cam.5.1.13575
Abstract | PDF

Filopodia are an important feature of actively motile cells, probing the pericellular environment for chemotactic factors and other molecular cues that enable and direct the movement of the cell. They also act as points of attachment to the extracellular matrix for the cell, generating tension that may act to pull the cell forward and/or stabilize the cell as it moves. Endothelial cell motility is a critical aspect of angiogenesis, but only a limited number of molecules have been identified as specific regulators of endothelial cell filopodia. Recent reports, however, provide evidence for the involvement of PECAM-1, an endothelial cell adhesion and signaling molecule, in the formation of endothelial cell filopodia. This commentary will focus on these studies and their suggestion that at least two PECAM-1-regulated pathways are involved in the processes that enable filopodial protrusions by endothelial cells. Developing a more complete understanding of the role of PECAM-1 in mediating various endothelial cell activities, such as the extension of filopodia, will be essential for exploiting the therapeutic potential of targeting PECAM-1.

Open Access Article

Neuronal migration illuminated: A look under the hood of the living neuron

Niraj Trivedi and David J. Solecki
Pages 42 - 47
http://dx.doi.org/10.4161/cam.5.1.13609
Abstract | PDF

During vertebrate brain development, migration of neurons from the germinal zones to their final laminar positions is essential to establish functional neural circuits^1-3. Whereas key insights into neuronal migration initially came from landmark studies identifying the genes mutated in human cortical malformations⁴, cell biology has recently greatly advanced our understanding of how cytoskeletal proteins and molecular motors drive the morphogenic cell movements that build the developing brain. This Commentary & View reviews recent studies examining the role of the molecular motors during neuronal migration and critically examines current models of acto-myosin function in the two-step neuronal migration cycle. Given the apparent emerging diversity of neuronal sub-type cytoskeletal organizations, we propose that two approaches must be taken to resolve differences between the current migration models: the mechanisms of radial and tangential migration must be compared and the loci of tension generation, migration substrates, and sites of adhesion dynamics must be precisely examined in an integrated manner.

Open Access Article

Cell adhesion molecules in context: CAM function depends on the neighborhood

Nicholas J. Gibson
Pages 48 - 51
http://dx.doi.org/10.4161/cam.5.1.13639
Abstract | PDF

Cell adhesion molecules (CAMs) are now known to mediate much more than adhesion between cells and between cells and the extracellular matrix. Work by many researchers has illuminated their roles in modulating activation of molecules such as receptor tyrosine kinases, with subsequent effects on cell survival, migration, and process extension. CAMs also are known to serve as substrates for proteases which can create diffusible fragments capable of signaling independently from the CAM. The diversity of interactions is further modulated by membrane rafts, which can co-localize or separate potential signaling partners to affect the likelihood of a given signaling pathway being activated. Given the ever-growing number of known CAMs and the fact that their heterophilic binding in cis or in trans can affect their interactions with other molecules, including membrane-bound receptors, one would predict a wide range of effects attributable to a particular CAM in a particular cell at a particular stage of development. The function(s) of a given CAM must therefore be considered in the context of the history of the cell expressing it and the repertoire of molecules expressed both by that cell and its neighbors.

Open Access Article

FAK: Dynamic integration of guidance signals at the growth cone

Mariola R. Chacón and Pietro Fazzari
Pages 52 - 55
http://dx.doi.org/10.4161/cam.5.1.13681
Abstract | PDF

During the formation of neural circuitry, axons are known to be guided to their specific targets by a relatively small arsenal of guidance signals. However, the molecular integration of this guidance information inside the axonal growth cone (GC) is still baffling. Focal adhesion kinase (FAK) is a cytosolic kinase which interacts with a complex molecular network via multiple phosphorylation sites. Paradoxically, FAK activation is required by both attractive and repulsive cues to control respectively axon outgrowth and disassembly of adhesive structures together with cytoskeletal dynamics. It was suggested that FAK might work as a versatile molecular integrator switching to different functions depending on its activation state. Two studies published recently by our group and Woo et al. shed light on this issue: for the first time, these works report a detailed molecular analysis of FAK activation and phosphorylation pattern in primary neuronal cultures in response to the repulsive cues Semaphorin3A and ephrinA1 respectively. Here we comment on the major novelties provided by these papers in the context of previous literature and we speculate on the future avenues of investigation opened by these works.




SHORT COMMUNICATIONS

Open Access Article

Movement within and movement beyond: Synaptotagmin-mediated vesicle fusion during chemotaxis

Richard A. Colvin and Andrew D. Luster
Pages 56 - 58
http://dx.doi.org/10.4161/cam.5.1.13197
Abstract | PDF

Leukocyte chemotaxis plays an essential role in generating and delivering immune responses and is a critical component of inflammation. In order to identify novel genes and pathways important for regulating chemotaxis, we performed an RNAi-based screen and identified several genes involved with vesicle movement and fusion as mediators of chemotaxis. Our recently published data¹ show that during chemotaxis vesicle trafficking proteins are required for lysosome fusion, uropod release and efficient directed cell migration.

Open Access Article

A model for phospho-caveolin-1-driven turnover of focal adhesions

Micha Nethe and Peter L. Hordijk
Pages 59 - 64
http://dx.doi.org/10.4161/cam.5.1.13702
Abstract | PDF

The regulation of Focal Adhesion (FA) dynamics is a key aspect of cellular motility. FAs concentrate integrins and associated cytoskeletal elements as well as a large number of regulatory proteins, including adapters, kinases and small GTPases of the Rho Family. We have recently shown that activated Rac1 can localize to FAs and can initiate the accumulation of the adapter protein Caveolin1 (Cav1) at FAs. As reported by several groups including ours, this translocation requires Cav1 phosphorylation at Tyr14, presumably by Src. Here we provide additional data regarding this process and briefly review recent literature. Finally, we incorporated the different pieces of available information into a mechanistic model. This model proposes that local Rac1 activation initiates a series of events that involve endosomal traffic of Cav1 and Src, targeting these proteins to or near FAs. Next, within specific membrane domains, Src can mediate the phosphorylation of Cav1 at Tyr 14, which is important for the stable FA localization of Cav1. Finally, dephosphorylation of Cav1 may represent a key step required for internalization, FA turnover and cell motility.




RESEARCH PAPERS

Open Access Article

Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Jennifer Forsprecher, Zhemeng Wang, Harvey A. Goldberg and Mari T. Kaartinen
Pages 65 - 72
http://dx.doi.org/10.4161/cam.5.1.13369
Abstract | PDF

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.

Open Access Article

Fibronectin-mediated upregulation of α5β1 integrin and cell adhesion during differentiation of mouse embryonic stem cells

Pimchanok Pimton, Saheli Sarkar, Nidhi Sheth, Anat Perets, Cezary Marcinkiewicz, Philip Lazarovici and Peter I. Lelkes
Pages 73 - 82
http://dx.doi.org/10.4161/cam.5.1.13704
Abstract | PDF

Embryonic stem (ES) cells have a broad potential application in regenerative medicine and can be differentiated into cells of all three germ layers. Adhesion of ES cells to extracellular matrix (ECM) proteins is essential for the differentiation pathway; Cell-ECM adhesion is mediated by integrins that have the ability to activate many intracellular signaling pathways. Therefore, we hypothesize that the expression and function of integrin receptors is a critical step in ES differentiation. Using functional cell adhesion assays, our study demonstrates that α5β1 is a major functional integrin receptor expressed on the cell surface of undifferentiated mouse ES-D3 cells, which showed significantly higher binding to fibronectin as compared to collagens. This adhesion was specific mediated by integrin α5β1 as evident from the inhibition with a disintegrin selective for this particular integrin. Differentiation of ES-D3 cells on fibronectin or on a collagen type1/fibronectin matrix, caused further selective up-regulation of the α5β1 integrin. Differentiation of the cells, as evaluated by immunofluorescence, FACS analysis and quantitative RT-PCR, was accompanied by the upregulation of mesenchymal (Flk1, isolectin B4, α-SMA, vimentin) and endodermal markers (FoxA2, SOX 17, cytokeratin) in parallel to increased expression of α5β1 integrin. Taken together, the data indicate that fibronectin-mediated, upregulation of α5β1 integrin and adhesion of ES-D3 cells to specific ECM molecules are linked to early stages of mouse embryonic stem cells commitment to meso-endodermal differentiation.

Open Access Article

Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns

Noelia Martinez-Molina, Yongsoo Kim, Philip Hockberger and Francis G. Szele
Pages 83 - 95
http://dx.doi.org/10.4161/cam.5.1.13788
Abstract | PDF

Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30-90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (>90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.