Journal: Nature Communications

Article Title: Salmonella exploits LRRK2-dependent plasma membrane dynamics to invade host cells

doi: 10.1038/s41467-025-57453-x

Figure Lengend Snippet: a and b Representative images ( a ) and quantifications ( b ) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells infected with the indicated S Tm strains. Cell lysates were collected and immunoblotted for phospho-RAB10 (T73), total RAB10, or ß-actin (loading control) at 30 min p.i. c and d , Representative images ( c ) and quantifications ( d ) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells infected with WT S Tm, treated with WT S Tm supernatant (STm S/N), dead WT S Tm, LPS (100 ng/ml) or Δ invA /Inv S Tm (invasion-deficient attenuated strain with inactivation of T3SS-1, and with invasin gene from Yersinia expressed) for 30 min. e and f Representative images ( e ) and quantifications of ( f ) phospho-RAB10 (T73) signal measured by western blot. WT, LRRK2 KO and TLR4 KO Henle 407 cells were infected with WT S Tm and cell lysates were collected at 10 min or 30 min p.i. and immunoblotted for TLR4, phospho-RAB10 (T73), total RAB10, or ß-actin (loading control). g and h Quantifications ( g ) and representative images ( h ) of phospho-RAB10 (T73) localization at WT S Tm invasion sites at the indicated times p.i. WT, LRRK2 KO or TLR4 KO Henle 407 cells were transfected with GFP-RAB10 and then infected with WT S Tm. Cells were fixed at 10 or 30 min p.i. and stained for phospho-RAB10 (T73) and S Tm. The cell boundaries in h are depicted by the white outlines. i WT Henle 407 cells and TLR4 KO Henle 407 cells were infected with WT S Tm and lysed at 2 hours p.i. for CFU counting. Data shown are means ± S.D. for three independent experiments. In g At least 100 invasion sites for each condition in each experiment were scored for the recruitment or the retention of RAB10. P values were calculated using one-way ANOVA ( b and d ), two-way ANOVA ( f and g ), or two tailed unpaired t -test ( i ). P values in d were calculated and labeled between the treated groups with the untreated control. Scale bars, 10 μm. Source data are provided as a Source Data file.

Article Snippet: Immunostaining was performed as previously described using the following primary antibodies: rabbit monoclonal anti-phospho-RAB10 T73 (Abcam, ab230261) at 1:100, mouse monoclonal anti-C-myc 9E10 (Thermo, MA1-980) at 1:500, rabbit polyclonal anti- Salmonella (BD Transduction, 229481) at 1:100, rabbit polyclonal anti-TLR4 (Novus Biologicals, NB100-56581) at 1:100, mouse monoclonal anti-PIEZO1 (Novus Biologicals, NBP2-75617) at 1:100 and chicken polyclonal anti-GFP (Rockland, 600-901-215S) at 1:500.

Techniques: Western Blot, Infection, Control, Transfection, Staining, Two Tailed Test, Labeling

Journal: Nature Communications

Article Title: Salmonella exploits LRRK2-dependent plasma membrane dynamics to invade host cells

doi: 10.1038/s41467-025-57453-x

Figure Lengend Snippet: a and b Representative images ( a ) and quantifications ( b ) of phospho-RAB10 (T73) and total RAB10 signal measured by western blot in WT Henle 407 cells treated with the PIEZO1 agonist, Yoda1. Cell lysates were collected and immunoblotted following a 30 min treatment with either DMSO or Yoda1. Data shown are means ± S.D. for three independent experiments. c and d Representative images ( c ) and quantifications ( d ) of phospho-RAB10 (T73) signal measured by western blot in WT Henle 407 cells. The cells were treated with the PIEZO1 inhibitor GsMTx4, LPS and/or the PIEZO1 agonist Yoda1, and cell lysates were collected and immunoblotted for phospho-RAB10 (T73), total RAB10, or ß-actin (loading control) following 30 min treatment. e Representative images of TLR4 and PIEZO1 localizations in uninfected condition. WT or TLR4 KO Henle 407 cells were transfected with GFP-RAB10, or RAB10 KO Henle 407 cells were nontransfected. Then the cells were fixed and stained for TLR4 and PIEZO1. f Representative images of TLR4 and PIEZO1 localizations in at 10 min p.i. WT, TLR4 KO or RAB10 KO Henle 407 cells were infected with WT S Tm and then fixed at 10 min p.i. and stained for S Tm, TLR4 and PIEZO1. The images in the ‘Merge’ channel represent the merged accumulative fluorescence signals from the TLR4, PIEZO1 and S Tm channels. g and h Quantifications of TLR4 ( g ) and PIEZO1 ( h ) localizations at invasion sites in ( f ). i WT Henle 407 cells were untreated or pretreated with indicated PIEZO1 inhibitors, and then infected with WT S Tm and lysed at 2 hours p.i. for CFU counting. At least 100 invasion sites ( g and h ) for each condition in each experiment were scored for the recruitment of TLR4 or PIEZO1. All images shown are representative images from three independent experiments. Data shown are means ± S.D. for three independent experiments. P values were calculated using one-way ANOVA. Scale bars, 10 μm. In e and f the cell boundaries are depicted by the white outlines. Source data are provided as a Source Data file.

Article Snippet: Immunostaining was performed as previously described using the following primary antibodies: rabbit monoclonal anti-phospho-RAB10 T73 (Abcam, ab230261) at 1:100, mouse monoclonal anti-C-myc 9E10 (Thermo, MA1-980) at 1:500, rabbit polyclonal anti- Salmonella (BD Transduction, 229481) at 1:100, rabbit polyclonal anti-TLR4 (Novus Biologicals, NB100-56581) at 1:100, mouse monoclonal anti-PIEZO1 (Novus Biologicals, NBP2-75617) at 1:100 and chicken polyclonal anti-GFP (Rockland, 600-901-215S) at 1:500.

Techniques: Western Blot, Control, Transfection, Staining, Infection, Fluorescence

Journal: Nature Communications

Article Title: Salmonella exploits LRRK2-dependent plasma membrane dynamics to invade host cells

doi: 10.1038/s41467-025-57453-x

Figure Lengend Snippet: a and b Representative images ( a ) and quantifications ( b ) of LRRK2 localization at invasion sites at 30 min p.i. WT or TMEM16F KO Henle 407 cells were transfected with LRRK2-mNeon and myc-PM-RAB10 and infected with WT or Δ sopD S Tm. Cells were fixed at 30 min p.i. and stained with myc-tag. Line plot profile follows respective white arrows in the insets of the ‘Merge’ channels on the left. c and d , Representative images ( c ) and quantifications of ( d ) phospho-LRRK2 (S935) signal measured by western blot. WT, LRRK2 KO and TLR4 KO Henle 407 cells were infected with WT S Tm and cell lysates were collected at 30 min p.i. and immunoblotted for phospho-LRRK2 (S935), total LRRK2, or ß-actin (loading control). Data shown are means ± S.D. for three independent experiments. At least 100 invasion sites for each condition in each experiment were scored for LRRK2 localization to S Tm invasion sites. P values were calculated using two-way ANOVA. Scale bars, 10 μm. Source data are provided as a Source Data file.

Article Snippet: Immunostaining was performed as previously described using the following primary antibodies: rabbit monoclonal anti-phospho-RAB10 T73 (Abcam, ab230261) at 1:100, mouse monoclonal anti-C-myc 9E10 (Thermo, MA1-980) at 1:500, rabbit polyclonal anti- Salmonella (BD Transduction, 229481) at 1:100, rabbit polyclonal anti-TLR4 (Novus Biologicals, NB100-56581) at 1:100, mouse monoclonal anti-PIEZO1 (Novus Biologicals, NBP2-75617) at 1:100 and chicken polyclonal anti-GFP (Rockland, 600-901-215S) at 1:500.

Techniques: Transfection, Infection, Staining, Western Blot, Control

Journal: Nature Communications

Article Title: Salmonella exploits LRRK2-dependent plasma membrane dynamics to invade host cells

doi: 10.1038/s41467-025-57453-x

Figure Lengend Snippet: a and b Representative images ( a ) and quantifications of ( b ) phospho-RAB10 (T73) signal measured by western blot. Henle 407 cells were infected with WT S Tm for 30 min and immunoblotted for indicated antibodies. c Representative images of Henle 407 cells transfected with GFP-RAB10 and DNM2-mCherry and infected with WT or Δ sopD S Tm. Cells were fixed at 30 min p.i. and stained for S Tm. Line plot profile follows respective white arrows in the insets of the ‘Merge’ channels on the left. d and e Quantifications of RAB10 ( d ) or DNM2 ( e ) localization at invasion sites at 30 min p.i. Data shown are means ± S.D. for three independent experiments. At least 100 invasion sites ( d and e ) for each condition in each experiment were scored for RAB10 or DNM2 localizations to invasion sites. P values were calculated using two-way ANOVA. Scale bars, 10 μm. Source data are provided as a Source Data file. f Model depicting the results of this study. Prior to infection, RAB10 is recruited to the plasma membrane (PM) where it stabilizes tubular invaginations (membrane reservoirs) . LRRK2 may also contribute to membrane tubulation through binding to acidic phospholipids, including phosphatidylserine (PS), on the cytoplasmic leaflet of the PM . RAB10’s T73 phosphorylation by LRRK2 maintains RAB10 in a GTP-bound state by preventing its interaction with GTPase activating proteins (GAPs) . In turn, phosphorylated RAB10 promotes LRRK2 activity at the membrane in a feed-forward activation loop . With S Tm infection, membrane reservoirs can be redistributed to S Tm invasion sites by carrier vesicle delivery and/or tubule resorption, helping to generate invasion ruffles . Phosphorylated and GTP-bound RAB10 is recruited to S Tm invasion sites and generates invaginated portions of the PM containing bacteria. Bacterial lipopolysaccharide (LPS) stimulates TLR4 to induce PIEZO1-mediated calcium influx , a signal that activates the PS scramblase TMEM16F . PS scrambling promotes LRRK2 release from the membrane, enabling RAB10 dephosphorylation by PPM1H and/or other phosphatases . Dephosphorylated RAB10 is targeted by SopD, a Salmonella T3SS effector with GAP activity . GDP-bound RAB10 interacts with DNM2 , promoting invaginated PM scission to generate SCVs .

Article Snippet: Immunostaining was performed as previously described using the following primary antibodies: rabbit monoclonal anti-phospho-RAB10 T73 (Abcam, ab230261) at 1:100, mouse monoclonal anti-C-myc 9E10 (Thermo, MA1-980) at 1:500, rabbit polyclonal anti- Salmonella (BD Transduction, 229481) at 1:100, rabbit polyclonal anti-TLR4 (Novus Biologicals, NB100-56581) at 1:100, mouse monoclonal anti-PIEZO1 (Novus Biologicals, NBP2-75617) at 1:100 and chicken polyclonal anti-GFP (Rockland, 600-901-215S) at 1:500.

Techniques: Western Blot, Infection, Transfection, Staining, Membrane, Binding Assay, Activity Assay, Activation Assay, Bacteria, De-Phosphorylation Assay