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: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (22 x optical sections of 0.173 μm) showing staining of GABAAR β3 in non-permeabilised 16-DIV hippocampal neurons (magenta). Cells co-express mRuby with NT-shRNA or GIT1shRNA, as indicated. Cell tracing was based on mRuby images. Cells were treated with 10 µM R18 for 24 h and 20 µM TAT or DJNKI-1 for 8 h. B. Quantification of GABAAR β3 enrichment in dendritic spines. R18 prevented DJNKI-1-induced GABAAR β3 increase at the dendritic spines. C. R18 prevented GIT1-S371A-induced increase in GABAAR β3 at the cell surface. Histogram bars represent means from ∼51 spines per condition from several neurons. P-values were calculated from Student’s two-tailed t-tests were compared to control (TAT) unless otherwise indicated. D. Neurons were co-transfected at 7 DIV with EGFP-Dynamin 2-WT or EGFP-Dynamin 2- K44A and treated with 20 µM TAT or DJNKI-1 for 8 h. Representative images are shown. E. Mean intensities for GABAAR β3 are shown from non-permeabilised cells in mushroom spines. GABAAR surface expression increased in DJNKI-1-treated neurons, even when the K44A mutant is expressed. Mean data +/- SEMs from ∼70 spines per condition are shown. p- values (calculated from Student’s two-tailed t test) were compared to control (TAT) unless otherwise indicated by brackets.

Article Snippet: For in vitro phosphorylation assay, pEBG-JNK1α1 and pEGFP-MEKK1Δ(1174-1493) have been previously described ( ). pmRuby2-Lifeact7 was from Michael Davidson’s lab via Addgene, pEGFP-Dynamin 2-WT and pEGFP-Dynamin 2-K44A was from Pietro De Camilli. pcDNA3-HA-Arf1 (Addgene plasmid # 10830) and pcDNA3-HA-Arf1-ActQ71L (Addgene plasmid # 10832) were generated by Thomas Roberts. pGEX-5x-1-VHS GAT-GGA3 was a gift from Juan S. Bonifacino. pEGFP-PXN-WT was described previously ( ).

Techniques: Staining, shRNA, Two Tailed Test, Control, Transfection, Expressing, Mutagenesis