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First published online April 16, 2004
doi: 10.1242/10.1242/jcs.01066

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Unconventional myosin VIIa and vezatin, two proteins crucial for Listeria entry into epithelial cells

¹ Unité des Interactions Bactéries-Cellules, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris CEDEX 15, France
² Unité de Génétique des Déficits Sensoriels, Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris CEDEX 15, France

View larger version (22K): [in a new window]   Fig. 1. Schematic representation of the myosin VIIa. The N-terminus of myosin VIIa consists of a conserved motor head containing the ATP- and actin-binding sites. The neck region, composed of five isoleucine-glutamine (IQ) motifs, is followed by a long tail of 1360 amino acids. The tail contains a short coiled-coil domain, which is implicated in the formation of homodimers, and two large repeats arranged in tandem separated by a putative SH3 domain. Each of these large repeats contains a MyTH4 and a FERM domain. The region interacting with vezatin is indicated.

View larger version (18K): [in a new window]   Fig. 2. Effect of BDM on Listeria and InlA-coated beads internalization. Entry of wild-type L. monocytogenes into human Caco-2 cells and of L. innocua expressing InlA and of InlA-coated beads into L2071 fibroblasts expressing human E-cadherin, in the presence of 10 mM or 50 mM BDM was evaluated and compared with entry in untreated cells. Vero cells were infected with wild-type L. monocytogenes and L. monocytogenes inlA in the absence or presence of BDM and the entry levels were determined. Entry to untreated cells has been normalized to 100 and the levels of entry in cells treated with BDM are expressed as relative values. Results are means±SD from three independent experiments, each done in triplicate.

View larger version (58K): [in a new window]   Fig. 3. Localization of the myosin VIIa tail fused to GFP in transfected Caco-2 cells. The myosin VIIa tail fused to GFP localizes with endogenous vezatin at the cell-cell contacts. Arrows show regions where GFP/myosin-VIIa-tail and vezatin colocalize.

View larger version (33K): [in a new window]   Fig. 4. Effect of myosin VIIa tail overexpression on the entry of InlA- and InlB-coated beads, L. monocytogenes inlA and inlB. (A) L2071 hEcad overexpressing the GFP/myosin-VIIa-tail were incubated with InlA- or InlB-coated beads. Arrows show sites where the GFP/myosin-VIIa-tail is recruited around entering InlA-coated beads. No recruitment was observed around InlB-coated beads. Boxed regions (left) are enlarged in the middle and on the right. The proportion of InlA- and InlB-coated beads associated with cells that recruit GFP/myosin-VIIa-tail was evaluated and compared with that of InlA-coated beads recruiting GFP alone. (B) L2071 hEcad were transfected with a plasmid expressing GFP alone (L2071 hEcad+GFP) or the tail of myosin VIIa fused to GFP (L2071 hEcad+ GFP-MyoVIIa tail). Entry values are expressed as proportion of intracellular beads or bacteria compared with the total number of beads or bacteria associated with the cells. Results are means±SD, from three independent experiments, each done in triplicate.

View larger version (121K): [in a new window]   Fig. 5. Recruitment of myosin VIIa and vezatin during entry of L. innocua expressing InlA into Caco-2 cells. (A) Actin and myosin VIIa labelling. Myosin VIIa is recruited and colocalizes with actin at the entry site of L. innocua expressing InlA and at the adherens junctions in cells not treated (0 BDM) or treated with 50 mM BDM. (B) Actin and vezatin labelling. Actin and vezatin colocalize at the entry site of Listeria and at the adherens junctions in untreated cells (0 BDM) or cells treated with 50 mM BDM. Scale bar, 3 µm.

View larger version (50K): [in a new window]   Fig. 6. Contribution of the C-terminus of -catenin in the recruitment of vezatin at adherens junction and in InlA-mediated entry. (A) Schematic representations of E-cadherin, -catenin and the chimeras fusing the extracellular domain of human E-cadherin (amino acids 1-580) to different regions of the C-terminal domain of -catenin and of the human E-cadherin variant lacking its cytoplasmic tail. Dotted lines indicate the regions of -catenin absent from each construct. (B) Expression level of each chimera in L2071 fibroblasts. Using the HECD1 antibody, the expression level of each chimera (defined by the fluorescence intensity of individual transfected cells) was measured for 150 transfected cells (50 transfected cells per coverslip) and compared with untransfected cells. Values are means±SD of three independent experiments. (C) Localization by fluorescence microscopy of vezatin and human E-cadherin in transfected L2071 cells expressing the different chimeras. (D) Proportion of intracellular beads and bacteria compared with total associated beads or bacteria in L2071 fibroblasts expressing the different chimeras. hEcad cells express the full-length human E-cadherin and hEcad-cyto cells express a variant of human E-cadherin lacking its cytoplasmic domain. Values are means±SD for three independent experiments, each done in triplicate.

View larger version (40K): [in a new window]   Fig. 7. Localization of myosin VIIa and uptake of InlA-coated beads and Listeria in cells expressing truncated forms of vezatin. (A) Schematic representation of vezatin variants fused to GFP. The transmembrane fragment (TM) of full-length vezatin (VezFL) is indicated in black. The region containing the myosin-VIIa-interacting site is indicated. Dotted lines indicate regions absent from each construct. (B) Expression level of each vezatin construct in L2071 hEcad fibroblasts. The fluorescence intensity of GFP was quantified in 150 transfected cells (50 cells per coverslip) for each vezatin-GFP construct and compared with that of untransfected cells. Values are means±SD of three experiments. (C) Localization by fluorescence microscopy of myosin VIIa and vezatin-GFP fusions in L2071 hEcad cells expressing the VezFL-GFP, VezC-ter and VezC-terT constructs. Arrows show sites where myosin VIIa and vezatin-GFP constructs are recruited around entering InlA-coated beads. (D) Proportion of intracellular beads and bacteria compared with the total number of associated beads or bacteria in L2071 hEcad cells expressing the different vezatin constructs. Values are means±SD of three experiments, each done in triplicate.

View larger version (40K): [in a new window]   Fig. 8. Model for InlA-dependent entry of Listeria into epithelial cells. Proteins known to play a role in entry are indicated, including E-cadherin, - and ß-catenins, vezatin, myosin VIIa, and actin. This model highlights how myosin VIIa could help the membrane rearrangements during Listeria entry.

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