gfp dynamin 2 k44a  (Addgene inc)

Journal: bioRxiv

Article Title: Ca 2+ and DRP1 drive endocytic lysosome reformation at tripartite contact sites

doi: 10.64898/2026.01.30.702748

Figure Lengend Snippet: (A) Immunoblot for the phosphorylation of mTOR effector proteins during different conditions. Torin was used as a positive control for mTOR inhibition. Tubulin was used as a loading control. (B) Quantification of the fold change p-S6K/S6K from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, **P= 0.0011 (Untreated VS Torin), ns P = 0.9809 (Untreated VS YM201636), ns P = 0.99997 (Untreated VS 15 min recovery), ns P = 0.6835 (Untreated VS 30 min recovery), ns P = 0.99997 (Untreated VS 60 min recovery). (C) Quantification of the fold change of p-S6K/S6K for from immunoblot data in (A). Data represents the mean of n=4 replicates. one-way ANOVA using Tukey’s multiple comparison, ***P = 0.000038 (Untreated VS Torin), ns P = 0.9370 (Untreated VS YM201636), ns P = 0.99999 (Untreated VS 15 min recovery), ns P = 0.999993 (Untreated VS 30 min recovery), ns P = 0.9605 (Untreated VS 60 min recovery). (D) Live-cell imaging of cells expressing LAMP1-GFP (green) stained with LysoTracker deep red (magenta) to assess the lysosomal reformation in presence of 250 nM torin. Images were acquired after 2 h YM201636 treatment and 30 min washout. (G) Average area of LAMP1-positive structures was quantified. Each dot represents the average area of per 100 µm 2 ROI; plot shows mean area from a total of 90 ROI from 30 cells across n=3 biological replicates; Unpaired t-test; ns, P = 0.1980 (DMSO vs Torin). (F) Live-cell imaging of cells expressing LAMP1-mScarlet (magenta) together with DNM2-GFP or the GTPase mutant DNM2 (K44A)-GFP to assess DNM2 function in tubule fission. Images were acquired after 2 h YM201636 treatment and 30 min. (G) Quantification of LAMP1-positive tubule length (magenta) in cells overexpressing wild-type or mutant DNM2. Each dot represents the length of a single tubule; plot shows mean length from a total of 150 tubules across n=3 biological replicates; Unpaired t-test; ns P = 0.0564 (DNM2 WT vs. DNM2 K44A). (H) Schematic representation of the proximity labelling assay using LAMP1 fused to the biotin ligase TurboID. (I) Volcano plot comparing the biotinylated proxisome of LAMP1-TiD in cells treated with YM201636 for 2 h plus 30 min recovery versus untreated controls. Proteins significantly enriched during tubulation and fission are highlighted on the right side of the plot. Data from n=3 biological replicates. (J) Major categories of candidate proteins enriched during tubulation and fission.

Article Snippet: The following commercially available plasmids were obtained: LAMP1-GFP (Cat. 34831/ Addgene), LAMP1-mScarlet (Cat. 98827/ Addgene), pSpCas9(BB)–2A-GFP (pX458) (Cat. 48138/ Addgene), pSpCAS9 (BB) 2A-puro (pX459) (Cat. 48139/ Addgene), EGFR-GFP (Cat. 32751/ Addgene), WT Dynamin 2-GFP (Cat. 34686/ Addgene), GFP-Dynamin2 K44A (Cat. 22301/ Addgene), BFP-KDEL (Cat. 49150/ Addgene), mCh-Climp63 (Cat. 136293/ Addgene), pmCherry C1 MFF (Cat. 157760/ Addgene), pCAG-mito-RCaMP1h (Cat. 105013/ Addgene).

Techniques: Western Blot, Phospho-proteomics, Positive Control, Inhibition, Control, Comparison, Live Cell Imaging, Expressing, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Caveolin-1 dependent regulation of cell-matrix interphase in 3D collagen gels

doi: 10.1101/2025.06.04.657900

Figure Lengend Snippet: (A) Representative Z stack MIP of WT MEFs in 1.0 mg/ml and 1.5 mg/ml 3D collagen gels treated with DMSO (control), Dynasore, Y27632 and both. Cell protrusions marked by a box shown as zoomed images. The graphs show the perimeter of the cells measured from Z stack images of DMSO (control), Dynasore, Y27632 and Dynasore+Y 27632 (Dyn+Y27) in 1.0 mg/ml (n=30,36,30 and 34, N=4) and 1.5 mg/ml (n=32,33,31 and 31, N=4) 3D collagen gels. (B) Representative Z stack MIP of Dyn K44A GFP transfected (or untransfected) WT MEFs (green) and labelled with Phalloidin (magenta) treated with DMSO (control) or Y27632 in 1.0 mg/ml and 1.5 mg/ml 3D collagen gels. Graphs show perimeter calculated from Z stack images of control (untransfected cells with DMSO), Dyn K44A (Dyn K44A GFP transfected cells with DMSO), Y 27632 (Untransfected cells with Y 27632) and Dyn K44A+Y27 (Dyn K44A GFP transfected cells with Y 27632) in 1.0 mg/ml (n=22,24,18 and 21, N=3) and 1.5 mg/ml (n=19,22,17 and 20, N=3) 3D collagen gels. (C) Representative cross-section images of WT MEFs on 2D glass coverslips treated with DMSO (control), Dynasore, Y 27632 and Dynasore+Y 27632 (Dyn+Y27), labelled with Phalloidin (green) and DAPI (blue). Graph shows the cell stiffness (Pa) (n=29,30,20 and 33, N=5). (D) Representative cross-section images of WT MEFs labelled with CTxB (GM1-CTxB) (green) and Transferrin (magenta) in 1.5 mg/ml 3D collagen gel. Cells were treated with DMSO (control), Dynasore, Y 27632 and Dynasore+Y 27632 (Dyn+Y27) treatment. The graphs show the percentage distribution profile of cells with visible endocytosis (black) or no-endocytosis (white). Graph shows percentage data for >100 cells counted in four individual experiments (N). (E, F) Representative Z stack MIP of WT MEFs treated with DMSO (control), Dynasore, Y27632 and Dynasore+Y27632 in (E) 1.0 mg/ml and (F) 1.5mg/ml 3D collagen gels labelled with Phalloidin (green) and reflectance (gray). The graphs show branch junctions and total branch number of (E) 1.0 mg/ml and (F) 1.5 mg/ml collagen gels (normalised to the volume of ROI). (E) 38, 44 and 42 images (n) for control, Dynasore and Dyn+Y27 respectively from 5 independent experiments (N) and 20 images (n) for Y 27632 from 3 independent experiments (N) and (F) 38, 40 and 38 images (n) for control, Dynasore and Dyn+Y27 respectively from 5 independent experiments (N) and 22 images (n) for Y 27632 from 3 independent experiments (N) has been analysed. Graphs show all data points with median and quarters and error bars map the spread of data points. Scale bar: 5 μm. Statistical analysis: One way ANOVA (*P<0.01, **P<0.001, ****P<0.00001).

Article Snippet: Dynamin-K44A-GFP was purchased from Addgene 22301.

Techniques: Control, Transfection

Journal: Research Square

Article Title: Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA

doi: 10.21203/rs.3.rs-3376558/v1

Figure Lengend Snippet: (A) Cos7 cells were transfected with GFP-Dyn2 WT or K44A (DN) and miRFP-670 LifeAct for 24 hours prior to infection with wild-type Chlamydia at MOI=20. Infection was monitored by live-cell confocal microscopy using a Nikon CSU-W1 spinning disk microscope, obtaining images every 20 seconds for 30 minutes to identify sites exhibiting actin and Dyn2 co-recruitment. Scale bar = 1 micron. (B) Schematic depicting Dyn2 oligomerization, highlighting disruption of Dyn2 scission by K44A mutation. (C) Mean fluorescence intensity (MFI) of Dyn2 recruitment at Chlamydia entry sites was quantified, normalized as percent maximal MFI, and plotted onto a line graph depicting %max Dyn2 MFI +/− SEM for each timepoint. Background Dyn2 fluorescence was subtracted prior to normalization, which was performed independently for each Dyn2 WT and DN recruitment event. (D) Kymographs depicting RFP-Dyn2, GFP- Chlamydia , and far red actin signal over a 30 minute timelapse. Top arrow indicates initiation of protein recruitment and bottom arrow indicates completion of pathogen entry. (E-G) Detailed analysis of each recruitment event obtained via live cell imaging, plotting the (E) internalization duration, (F) rate of Dyn2 recruitment and (G) Dyn2 turnover of each event on a violin plot with inset boxplot reporting the median value +/− SD for each condition. (E) Internalization duration was quantified by calculating the elapsed time between initiation of protein recruitment and termination of pathogen entry, as detailed in Fig. S1. Individual rates of Dyn2 recruitment (F) and turnover (G) were calculated by measuring the slope derived from basal Dyn2 MFI to peak MFI for recruitment, and peak Dyn2 MFI to basal MFI for turnover, as detailed in Fig. S1. Data was obtained from a minimum N=23 individual rates. Statistical significance was determined by Wilcoxon ranked-sum. All data are representative of 3 independent experiments, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Dyn2-pmCherryN1 was a gift from Christien Merrifield (Addgene plasmid #27689), RFP Dynamin2 K44A was a gift from Jennifer Lippincott-Schwartz (Addgene plasmid #128153), WT Dyn2 pEGFP was a gift from Sandra Schmid (Addgene plasmid #34686), and GFP-Dynamin 2 K44A was a gift from Pietro De Camilli (Addgene plasmid #22301).

Techniques: Transfection, Infection, Confocal Microscopy, Microscopy, Disruption, Mutagenesis, Fluorescence, Live Cell Imaging, Derivative Assay