BSCB Newsletter, Winter 2005

BSCB Autumn Meeting
Heriot Watt University, 2-5 October 2005

An unseasonably sunny Scotland greeted more than 80 participants from the UK, Europe and the US., who gathered for the Society's autumn meeting on 'Signalling and cytoskeletal dynamics during infection' in the Edinburgh Conference Centre at Heriot Watt University. The organiser, Michael Way (Cancer Research UK), had assembled an enticing programme of cutting-edge talks from leading experts studying cytoskeletal manipulation by bacteria, parasites and viruses.

By Richard D. Hayward

The plenary lecture on Sunday evening certainly lived up to this expectation. Pascale Cossart (Pasteur Institute, France) eloquently summarised two decades of work using genetics, post-genomics and cell biology to decipher how the food-borne bacterium Listeria manipulates the actin cytoskeleton of the host cell to force its own internalisation, actin-based motility and intercellular spread. Studying the subversive actions of the underlying virulence factors has not only enhanced understanding of listeriosis, but also contributed significantly to deciphering many fundamental cellular processes. Epitomising this, the most recent data from her laboratory demonstrate that by binding the host receptor E-cadherin, Listeria hijack the clathrin-dependent endocytic machinery to enter cells. These experiments also illuminated a novel cell-cell junction component (ARHGAP10) that controls the recruitment of a-catenin, a central linker between transmembrane receptors and the actin cytoskeleton.

Ari Helenius (Swiss Federal Institute of Technology, Switzerland) began the first session on Invasion by describing how many animal viruses, like adenovirus, influenza and SV40, differentially harness the cellular endocytic pathway to guide them through a complex entry and uncoating programme. Viruses can therefore be employed as tools to probe plasma membrane dynamics and membrane fusion and to define endocytic routes. To illustrate this, he showed how SV40 infection can be used to assess the specific dynamics, activation and assembly of cholesterol-rich caveolae. The obligate intracellular parasite Toxoplasma gondii also infects vertebrate cells, but by gliding, a phenotype that requires the collaborative action of secretory adhesins, molecular motors and factors involved in actin polymerisation. This gliding motility is powered by myosin A and requires actin filaments, but how the parasite controls actin polymerisation is unknown.

Dominique Soldati (University of Geneva, Switzerland) showed that apicomplexan genomes encode proteins homologous to eukaryotic actin regulatory proteins, in particular profilin- and formin-like proteins that contribute to this essential invasive process.  John Leong (University of Massachusetts Medical School, USA) described how extracellular enterohaemorrhagic E. coli (EHEC) utilise a specialised 'type III' secretion system (TTSS) to deliver effector proteins into mammalian cells that reorganise the actin cytoskeleton into adhesion pedestals. One essential effector, Tir, inserts into the target plasma membrane and acts as a receptor for the bacterial surface protein intimin. He described exciting recent work in his laboratory showing that EHEC also translocates a second critical effector EspFU (also known as TccP) that mediates recruitment of the cellular actin assembly machinery to Tir. He presented the latest insights into the mechanism of EspFU and its co-operation with the cellular nucleation-promoting factor N-WASP, and reported apparent parallels between EHEC EspFU-dependent actin polymerisation and an ancestral auxiliary pathway employed by related enteropathogenic E. coli, also recently characterised by his group.

Freddy Frischknecht (University of Heidelberg, Germany) showed remarkable images of Plasmodium sporozoites within mosquito salivary glands, during injection, and inside the mammalian host. This in vivo imaging revealed unexpectedly that malarial sporozoites invade the lymphatic system and interact with a surprisingly diverse repertoire of cell types. Although highly elastic in vivo, the crescent-like sporozoites move only in concentric circles in vitro, suggesting they might also provide a tractable experimental system with which to study cellular motility.

The dynamic organisation of the plasma membrane and its receptors remains an outstanding puzzle in cell biology. Urs Greber (University of Zurich, Switzerland), who chaired the session on 'Signalling at the plasma membrane and beyond', demonstrated how labelled adenovirus can be employed to monitor transmembrane receptor dynamics using total internal reflection fluorescence microscopy and rapid-rate imaging. Different motility modes could be classified and modelled, including confined motion due to receptor interaction, and both slow directional and fast processive movements of activated integrins. The bacterial enteropathogen Salmonella delivers a cocktail of effectors into target cells via a secretion system similar to that encoded by EHEC.  These manipulate actin dynamics and force bacterial internalisation into intestinal epithelial cells.

Vassilis Koronakis (University of Cambridge) showed that effector delivery requires plasma membrane cholesterol, which binds to a conserved integral membrane TTSS translocon component. He also described how two Salmonella actin-binding proteins, SipC and SipA, co-operate to nucleate actin polymerisation independently of cellular components and stabilise the generated filaments by preventing their depolymerisation by host ADF/cofilin and gelsolin. New findings also provided insights into interplay between these Salmonella actin-binding proteins and other effectors that mimic cellular Rho GTPase nucleotide exchange factors and an inositol polyphosphatase.

In a related short talk, Markus Schlumberger (ETH Zurich, Institute of Microbiology, Switzerland) presented his recent work in Wolf-Dietrich Hardt's laboratory using fluorescent fusion proteins to monitor the delivery of Salmonella SipA in real-time, which was also awarded one of the poster prizes generously contributed by Cellular Microbiology.

Peter Cullen (University of Bristol) described sorting nexins, a large family of phox homology (PX) domain-containing proteins that regulate cargo sorting within the early endosomal network. In particular, he highlighted how modular sorting nexin-1 (SNX1) combines PX and a Bin/Amphiphysin/Rvs domain to associate with high curvature phosphatidylinositol-3-phosphate-rich early endosomal compartments, which also resemble Salmonella-containing vacuoles (SCVs). After dinner in the Scholar's Restaurant, the poster session was vivacious, promoting lively debate and interdisciplinary exchange of ideas, fuelled of course by the sponsored drinks.

The Tuesday morning session 'Cytoskeleton and signalling during replication' began with David Holden (Imperial College London) describing the characterisation of a second TTSS in Salmonella using signature-tagged mutagenesis. This system controls bacterial replication and survival by delivering effector proteins across the vacuolar membrane which manipulate SCV biogenesis. Recent work in his laboratory shows that two effectors, SifA and SseJ, and microtubule motors co-ordinately influence SCV morphology, while SseG is required to localise SCVs in proximity to the Golgi, facilitating interactions between SCVs and the secretory pathway which promote pathogen replication.

Michael Way related how vaccinia virus is able to hijack the cytoskeleton of the cell at multiple stages of its lifecycle. Vaccinia utilise not only actin-based motility as do Listeria, but also microtubule-dependent movement and  they can also induce cell motility and the formation of membrane extensions. He described exciting new data connected with the latter events, which reveal how the virus interferes with Rho GTPase regulation. Kristen Hager (University of Notre Dame, Indiana, USA) presented compelling evidence to support the hypothesis that Toxoplasma gondii uses g-tubulin as a scaffold to remodel the host cytoskeleton to generate a protective intracellular niche that nevertheless permits selective nutrient delivery. Phagosome-associated actin might also fulfil such a role, by providing tracks to guide intracellular compartments like lysosomes to dock with phagosomes prior to their fusion.

Gareth Griffiths (EMBL Heidelberg, Germany) described with characteristic passion the evidence supporting this premise based on work with an in vitro system established in his laboratory, which uses latex bead phagosomes to monitor actin assembly on the phagosome membrane. He additionally presented recent findings supporting a role for pro-inflammatory signalling lipids in assembly.

In 'Getting to the cell periphery', Greg Smith (Northwestern University Medical School, Chicago, USA) showed how live-cell fluorescence microscopy of herpesviruses has revealed that anterograde and retrograde particle transport along microtubules in sensory axons is accompanied by changes in viral particle composition. Tim Newsome (Cancer Research UK), a postdoctoral fellow in Michael Way's laboratory, described how cellular tyrosine kinases regulate vaccinia virus transport. Intracellular vaccinia initially traverse the microtubule network to reach the plasma membrane by recruiting the kinesin motor complex. This interaction is regulated by Src-family kinases, which upon activation by the viral B5R protein, phosphorylate the viral protein A36R. This switch triggers kinesin dissociation and induces actin-dependent propulsion of cell-associated extracellular virus particles, assisting intercellular spread.

Matt Welch (University of California Berkeley, USA) described new model systems to investigate mechanisms of cellular actin polymerisation. He discussed how the bacterial pathogen Rickettsia is able to induce actin polymerisation by recruiting and activating the Arp2/3 complex using a bacterial nucleation promoting factor called RickA, and how baculoviruses induce actin polymerisation both in the infected cell cytosol and within the nucleus late in infection.

The morning after a night of intensive ceilidhing, the final session considered 'Exit at the plasma membrane'. Daniel Kalman (Emory University, Atlanta, USA) described work on Abl and Src family kinases involved in the pathogenesis of infectious microbes. He showed that motility and release of vaccinia virus and adhesion pedestal assembly by enteropathogenic E. coli depend on these kinases, and outlined recent work on kinase recruitment by Bettina Bommarius in his laboratory, which also appeared on her prize-winning poster. The prospects for applying kinase inhibitors like Gleevec as an alternative to conventional drugs were discussed.

Guy Tran Van Nhieu (Pasteur Institute, Paris) summarised how Shigella utilise TTSS-dependent effector delivery to trigger their internalisation by synergistically stimulating host Rho GTPases and Src tyrosine kinase. He described how ATP release through connexin channels enhances Shigella invasion and intercellular spread, and a fluorescence-based method to directly visualise translocated bacterial effectors within target cells.

Having donned our 3D glasses, we were guided through the fascinating ultrastructure of a poxvirus infected cell by John Heuser (St. Louis, USA), including a detailed examination of the membrane and cytoskeletal alterations that accompany membrane fusion, and the formation of the actin 'comet tails' that allow extracellular virions to 'surf' on the infected cell surface.

Right: Looking down onto the intact surface of a cultured cell infected with vaccinia virus. Viral progeny on the exterior induce intracellular sub-membranous actin polymerisation, which creates membrane ridges behind the attached particles, allowing them to ‘surf’ across the cell surface to seek out a new target. Micrograph courtesy of John Heuser.

Quentin Sattentau (Oxford University) described the assembly of the 'virological synapse' by human immunodeficiency virus type-1, which requires viral envelope glycoprotein receptor engagement, associated adhesion molecules and the actin and microtubule cytoskeleton. These structures apparently allow communication between infected and uninfected CD4+ T cells, so might be central to cell-to-cell spread.

The relatively small size of this meeting fostered many productive discussions between the invited speakers, postdoctoral researchers and graduate students. Sincere thanks should go to Michael Way for organising such a scientifically productive and socially enjoyable meeting.

This report is merely intended to provide a flavour of the meeting. Owing to space constraints, it has not been possible to detail every presentation and poster. Apologies to those whose work has not been referred to directly.

Richard D. Hayward
Department of Pathology
University of Cambridge
rdh24@mole.bio.cam.ac.uk

The meeting was sponsored by Science International AAS, PeproTech EC Ltd., Media Cybernetics U.K., Li-Cor Biosciences Ltd., Cancer Research UK, Company of Biologists, EMBO and The Royal Microscopical Society.