Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails-dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, form-in-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.Fil: Surtayeva, Sofya. University of Connecticut School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velle, Katrina B.. University of Connecticut; Estados UnidosFil: Campellone, Kenneth G.. University of Connecticut; Estados UnidosFil: Talman, Arthur. Yale School of Medicine; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agaisse, Hervé. Yale School of Medicine; Estados UnidosFil: Wu, Yi I.. University of Connecticut School of Medicine; Estados UnidosFil: Loew, Leslie M.. University of Connecticut School of Medicine; Estados UnidosFil: Mayer, Bruce J.. University of Connecticut School of Medicine; Estados Unido

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin-based motility

Vaccinia virus dissemination relies on the N-WASP– ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP– ARP2/3 and Rac1–FHOD1 pathways.Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Yale. School of Medicine; Estados UnidosFil: Agaisse, Herve. University of Yale. School of Medicine; Estados Unido

The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to ER-Chlamydia Inclusion Membrane Contact Sites

Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development

Evolutionary Perspectives on the Moonlighting Functions of Bacterial Factors That Support Actin-Based Motility

Various bacterial pathogens display an intracellular lifestyle and spread from cell to cell through actin-based motility (ABM). ABM requires actin polymerization at the bacterial pole and is mediated by the expression of bacterial factors that hijack the host cell actin nucleation machinery or exhibit intrinsic actin nucleation properties.Various bacterial pathogens display an intracellular lifestyle and spread from cell to cell through actin-based motility (ABM). ABM requires actin polymerization at the bacterial pole and is mediated by the expression of bacterial factors that hijack the host cell actin nucleation machinery or exhibit intrinsic actin nucleation properties. It is increasingly recognized that bacterial ABM factors, in addition to having a crucial task during the intracellular phase of infection, display “moonlighting” adhesin functions, such as bacterial aggregation, biofilm formation, and host cell adhesion/invasion. Here, we review our current knowledge of ABM factors and their additional functions, and we propose that intracellular ABM functions have evolved from ancestral, extracellular adhesin functions

The metalloprotease Mpl supports Listeria monocytogenes dissemination through resolution of membrane protrusions into vacuoles

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the plcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔctA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wildtype actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution.Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Agaisse, Hervé. University of Virginia; Estados Unido

A <em>C. trachomatis</em> Cloning Vector and the Generation of <em>C. trachomatis</em> Strains Expressing Fluorescent Proteins under the Control of a <em>C. trachomatis</em> Promoter

<div><p>Here we describe a versatile cloning vector for conducting genetic experiments in <i>C. trachomatis</i>. We successfully expressed various fluorescent proteins (i.e. GFP, mCherry and CFP) from <i>C. trachomatis</i> regulatory elements (i.e. the promoter and terminator of the <i>incDEFG</i> operon) and showed that the transformed strains produced wild type amounts of infectious particles and recapitulated major features of the <i>C. trachomatis</i> developmental cycle. <i>C. trachomatis</i> strains expressing fluorescent proteins are valuable tools for studying the <i>C. trachomatis</i> developmental cycle. For instance, we show the feasibility of investigating the dynamics of inclusion fusion and interaction with host proteins and organelles by time-lapse video microscopy.</p> </div

Time-lapse video microscopy of <i>C. trachomatis</i> inclusion fusion.

<p>Selected merged frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057090#pone.0057090.s010" target="_blank">Video S2</a> acquired every 20 minutes by time-lapse video microscopy of HeLa cells co-infected with <i>C. trachomatis</i> strains expressing GFP (green) or mCherry (red) under the control of the <i>incD</i> promoter. The first frame corresponds to 24 h post infection. For each time point, an XY view (Top panels, XY) and an extended focus view (Bottom Panels, Ext.Focus) are shown. The time (hours: minutes) is indicated in the upper right corner of each frame. Scale Bar: 6 µm.</p

The CAD domain of STIM1 is required for STIM1 localization to ER-Inclusion MCSs.

<p>A. Schematic representation of the major domains of the STIM1 protein, their respective amino acid residue position and their respective cellular localization (ER lumen or Cytosol). Signal: signal peptide; EF: EF-hand; SAM: sterile alpha motif; TM: transmembrane domain; CC1: coiled-coil 1; CC2: coiled-coil 2; CAD: CRAC activation domain; S/P: Serine-proline-rich region; K: lysine-rich region. B. Confocal micrographs of HeLa cells expressing the indicated mCherry-STIM1 construct (red) and infected with <i>C</i>. <i>trachomatis</i> for 24h. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion and N indicates the nucleus. Scale bar: 10μm. C. Quantification of inclusion association of the indicated mCh-STIM1 constructs compared to full-length mCh-STIM1. *** p value <0.001. D. Confocal micrographs of HeLa cells expressing YFP-CAD (CAD(342–448), yellow), and infected for 24h with a strain of <i>C</i>. <i>trachomatis</i> expressing CFP (cyan). The merge is shown on the right. Two representative examples of YFP-CAD pattern on the inclusion are shown. The top and bottom panels respectively correspond to the extended focus view combining all the confocal planes (Ext.Foc.) and a single plane crossing the middle of the inclusion (XY View). The asterisk in the XY View Merge panel indicates the inclusion. Scale bar: 10μm.</p