dynamin 2 mcherry  (Addgene inc)

Journal: Cell reports

Article Title: Apical clathrin-coated pits control the location, timing, and scale of microvillar growth

doi: 10.1016/j.celrep.2025.116008

Figure Lengend Snippet: (A) Scanning electron microscopy (SEM) of native mouse small intestine along the full crypt-villus axis. Scale bar: 50 μm. (B) High magnification of the small intestinal crypt (scale bar: 10 μm) with magnification (base, middle, and transition [scale bars: 1 μm for solid box magnification and 500 nm for dashed box magnification]) showing the accumulation of microvilli along the transit-amplifying zone. (C) Native mouse small intestine stained for Villin, Dynamin2, Pacsin2, and clathrin light chain A (CLTA). Shown are single z plane confocal images of intestinal crypts. Scale bar: 10 μm. (D) Fold change in mean brush border intensity measurements for each marker from the base of the crypt relative to the crypt-villus transition zone. n = 20–25 crypts per marker, 3 wild-type mice. Kruskal-Wallis test with multiple comparisons; Pacsin2 *** p = 0.0007. CLTA *** p = 0.0003.

Article Snippet: DNMII-pmCherry-N1 (dynamin2) (human) , Addgene , #27689.

Techniques: Electron Microscopy, Staining, Marker

Journal: Cell reports

Article Title: Apical clathrin-coated pits control the location, timing, and scale of microvillar growth

doi: 10.1016/j.celrep.2025.116008

Figure Lengend Snippet: (A and C) Montage of a single microvillus growth event in CL4 cells expressing mCherry-espin and (A) pPLC-ΔPH-GFP (PIP(4,5)P 2 marker) and (C) EGFP-CLTA. Every column represents 30 s. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (B and D) Average fluorescence intensity measurements over time. (B) n = 10 growth events from 6 cells across 3 independent experiments. (D) n = 21 growth events from 8 cells across 3 independent experiments. (E) Montage of a single microvillus growth event in CL4 cells expressing mCherry-espin and EGFP-Pacsin2. Each column represents 1 min. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (F) Average fluorescence intensity measurements over time. n = 20 growth events from 10 cells across 3 independent experiments. (G) Montage of a single microvillus growth event in CL4 cells expressing mCherry-Dynamin2 and GFP-espin. Every column represents 1 min. Arrows mark the analyzed core bundle. Column width = 2.09 μm. (H) Average fluorescence intensity measurements over time. n = 10 growth events from 4 cells across 3 independent experiments.

Article Snippet: DNMII-pmCherry-N1 (dynamin2) (human) , Addgene , #27689.

Techniques: Expressing, Marker, 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 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: The DNM2-mCherry construct was a gift from Christien Merrifield (Addgene plasmid #27689) and was described previously .

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