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Kaniuk, Natalia A.,Canadien, Veronica,Bagshaw, Richard D.,Bakowski, Malina,Braun, Virginie,Landekic, Marija,Mitra, Shuvadeep,Huang, Ju,Heo, Won Do,Meyer, Tobias,Pelletier, Laurence,Andrews‐,Poly Blackwell Publishing Ltd 2011 Cellular microbiology Vol.13 No.11
<P><B>Summary</B></P><P>The facultative intracellular pathogen <I>Salmonella enterica</I> serovar Typhimurium establishes a replicative niche, the <I>Salmonella</I>‐containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B<SUP>+</SUP> tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1<SUP>+</SUP> tubules (the <I>Salmonella</I>‐induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin‐1. Here we find that Arl8B is required for kinesin‐1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell‐to‐cell transfer to infect neighbouring cells, and that both phenotypes require kinesin‐1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell‐to‐cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co‐opted by <I>S</I>. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.</P>
Badyal, Sandip K.,Basran, Jaswir,Bhanji, Nina,Kim, Ju Hwan,Chavda, Alap P.,Jung, Hyun Suk,Craig, Roger,Elliott, Paul R.,Irvine, Andrew F.,Barsukov, Igor L.,Kriajevska, Marina,Bagshaw, Clive R. Elsevier 2011 Journal of molecular biology Vol.405 No.4
<P>The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca<SUP>2+</SUP> binds to the EF2 domain of S100A4 with micromolar affinity and that the <I>K</I><SUB>d</SUB> value for Ca<SUP>2+</SUP> is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in <I>K</I><SUB>d</SUB> results from a reduced dissociation rate constant (from 16 s<SUP>− 1</SUP> to 0.3 s<SUP>− 1</SUP> in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these <I>in vitro</I> data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca<SUP>2+</SUP>]. However, for Ca<SUP>2+</SUP> transients to be effective in further promoting dissociation, the elevated Ca<SUP>2+</SUP> signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with <I>in vitro</I> data.</P>