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First published online 17 February 2004
doi: 10.1242/jcs.00949

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Microtubule motors control membrane dynamics of Salmonella-containing vacuoles

Julie Guignot^1,*, Emmanuelle Caron², Carmen Beuzón³, Cecilia Bucci⁴, Jonathan Kagan⁵, Craig Roy⁵ and David W. Holden^1,

¹ Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, London, SW7 2AZ, UK
² Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College London, London, SW7 2AZ, UK
³ Departamento de Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus de Teatinos, Málaga E-29071, Spain
⁴ Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università di Lecce, Via Monteroni, 73100 Lecce, Italy
⁵ Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA

View larger version (56K): [in a new window]   Fig. 1. Remodelling of the microtubule network by intracellular S. typhimurium. Cells were infected for 10 hours with S. typhimurium and fixed with cold methanol. Bacteria (WT, green in merged images) were detected with an anti-LPS antibody and cytoskeletal proteins (red in merged images) were detected using either an antibody against ß-tubulin, or TRITC-phalloidin to visualize F-actin. (A) Confocal x/y plane micrographs showing tubulin accumulation around intracellular bacteria in HeLa cells (upper panel), murine elicited peritoneal macrophages (middle panel) and INT407 cells (lower panel). (B) x/z section generated from a confocal z-stack of an infected HeLa cell. (C) Confocal micrographs of representative cells treated with cytochalasin D (cyto D) (lower panel) or nocodazole (noc) (upper panel) showing F-actin and tubulin (red in merged images) around S. typhimurium (green in merged images). Scale bars: 5 µm. Dotted lines indicate cell perimeters.

View larger version (47K): [in a new window]   Fig. 2. S. typhimurium mediates microtubule remodelling. (A) HeLa cells were transfected with a vector expressing FcRIIA, exposed to IgG-opsonized latex beads, then fixed 10 hours later and immunolabelled for ß–tubulin. Arrow indicates position of a latex bead. Scale bar corresponds to 10 µm. (B) Bacterial protein synthesis was blocked by adding tetracycline (Tet) at 7 hours p.i. Cells were fixed at 12 hours p.i, and examined for tubulin distribution. Results in left panel show the means ± s.d. of three independent experiments in which at least 70 S. typhimurium microcolonies were examined. Confocal images in right panel show tubulin distribution (red) around an S. typhimurium microcolony (green) in untreated and tetracycline-treated HeLa cells. (C) Representative confocal micrographs showing tubulin distribution in cells infected with S. typhimurium mutant strains. HeLa cells were infected for 8 hours with a sifA mutant or 10 hours for SPI-2 (ssaV–), SPI-1 (prgH–) or SPI-1/SPI-2 (prgH–, ssaV–) null mutant strains (green in merged images). Tubulin distribution (red in merged images) was examined by confocal microscopy. More than 50 S. typhimurium microcolonies containing at least three bacteria were examined for each strain. Scale bar: 5 µm. Dotted lines indicate cell perimeters.

View larger version (33K): [in a new window]   Fig. 3. Kinesin and dynein are required for S. typhimurium replication in HeLa cells. (A) Confocal images of dynein and kinesin distribution (red in merged images) in uninfected cells and cells infected for 10 hours with S. typhimurium (WT, green in merged images). Scale bar: 5 µm. Broken lines indicate cell perimeters. (B) Bacterial replication in p50/dynamitin-transfected cells (p50), KLC2-TPR transfected cells (TPR) or in p50/dynamitin, KLC2-TPR co-transfected cells (p50+TPR), was investigated by counting the number of S. typhimurium per infected cell. Results shown are the means ± s.d. of at least three independent experiments in which a total of at least 100 bacteria were examined. (C) Replication of S. typhimurium in the presence or absence of aurintricarboxilic acid (ATA). The values indicate the fold increase in bacterial strain cfu's between 2 hours and 16 hours after bacterial entry, relative to that of the wild-type strain, and represent the means ± s.d. of at least three independent experiments. The ssaV mutant serves as a control to detect impaired replication of bacteria. (D) Microtubule motors are required for Sif formation. Cells were infected with S. typhimurium fixed at 8 hours p.i. and labelled with an anti-LAMP-1 antibody in nocodazole-treated (noc), p50/dynamitin-transfected (p50), KLC2-TPR-transfected (TPR), or aurintricarboxilic acid (ATA)-treated cells to quantify Sif formation by confocal microscopy. Results shown are the mean ± s.d. of at least three independent experiments in which more than 100 infected cells were analysed for each experiment.

View larger version (37K): [in a new window]   Fig. 4. Effect of inhibiting kinesin or dynein on SCVs. HeLa cells were treated with aurintricarboxilic acid (ATA) 3 hours prior to infection, or transfected with vectors expressing p50/dynamitin (p50) or GFP-KLC2-TPR (TPR) 16 hours prior to infection with either wild-type (WT) S. typhimurium or a sifA mutant strain (green in merged images). Cells were fixed 8 hours p.i. and labelled with an anti-LAMP-1 antibody (red in merged images). (A) Representative confocal micrographs showing LAMP-1 distribution in cells infected with GFP-WT bacteria. Left panel of p50-expressing cells is at lower magnification to show redistribution of LAMP-1 to cell periphery (indicated by arrowheads); boxed area is at higher magnification. Right panel of p50-expressing cells shows a second example of SCVs at higher magnification. Scale bar: 2 µm. (B) Representative confocal micrographs showing LAMP-1 distribution (red in merged images) in cells infected with GFP-sifA mutant (green in merged images). (C) Association of bacteria with LAMP-1 in untreated cells or cells expressing p50 or TPR, or which had been incubated with ATA. Association was evaluated by confocal microscopy. Results show the mean ± s.d. of at least three independent experiments in which more than 100 bacteria were analysed for each experiment.

View larger version (34K): [in a new window]   Fig. 5. Inhibition of Rab7 affects the SCV membrane. HeLa cells were transfected with vectors encoding GFP-tagged wild-type Rab7, Rab7-T22N (dominant negative variant), or RILP-C33 (truncated Rab7-binding variant of RILP) prior to infection with S. typhimurium. (A) Representative confocal micrographs showing LAMP-1 distribution (red in merged images) in GFP-Rab7-, GFP-Rab7-T22N-, and GFP-RILP-C33-expressing cells infected for 8 hours by GFP-S. typhimurium (WT) or GFP-sifA mutant strains (bacteria are green in merged images, GFP-tagged Rab7, Rab7-T22N and RILP are not visible because of significantly lower overall fluorescence intensity). Scale bar: 5 µm. (B) LAMP-1 association with bacteria in HeLa cells infected for 8 hours with GFP-S. typhimurium (white bars) or the GFP-sifA mutant (grey bars). LAMP-1 association was assessed by confocal microscopy. Results shown are the mean ± s.d. of at least three independent experiments in which more than 100 bacteria were analysed for each experiment.

View larger version (69K): [in a new window]   Fig. 6. Inhibition of Rab7 prevents dynein recruitment to SCVs. Representative confocal micrographs showing dynein distribution (red in merged images) in GFP-Rab7-(upper panel), GFP-Rab7-T22N-(middle panel), or GFP-RILP-C33-expressing cells (lower panel) infected with S. typhimurium (WT, green in merged images) for 10 hours. Vacuolar membrane integrity was assessed by labelling LAMP-1 (blue in merged images). Scale bar: 5 µm.

View larger version (34K): [in a new window]   Fig. 7. RILP recruitment to SCVs is controlled by Rab7. (A) Representative confocal micrographs showing distribution of overexpressed GFP-RILP (green in merged image) in an uninfected cell (upper panel) or one infected with S. typhimurium (WT, red in merged image) for 10 hours (lower panel). Scale bar: 5 µm. Dotted lines indicate cell perimeter. (B) Representative confocal micrographs showing distribution of endogenous RILP (red in merged images) in an untransfected cell (upper panel) and a Rab7-T22N-expressing cell (lower panel), infected with S. typhimurium (green in merged images) for 10 hours. Vacuolar membrane integrity was assessed by labelling LAMP-1 (blue in merged images). Scale bar: 2 µm. (C) LAMP-1 association with bacteria in HeLa cells infected for 8 hours with S. typhimurium (white bars) or the sifA mutant (grey bars) in cells overexpressing GFP-RILP or GFP-RILP together with myc-Rab7-T22N. LAMP-1 association with bacteria was assessed by confocal microscopy. Results shown are the mean ± s.d. of at least three independent experiments in which more than 100 bacteria were analysed for each experiment.