Journal: Journal of Cell Science

Article Title: Borg5 restricts contractility and motility in epithelial MDCK cells

doi: 10.1242/jcs.261705

Figure Lengend Snippet: Borg5-depleted cells have increased E-cadherin-mediated cell–cell adhesion tension, undergo collective streaming and are less resistant to mechanical trituration. (A,B) FRET efficiency of the E-cadherin tension sensor (EcadTSMod) at cell–cell contacts in Borg5KD or Borg5KD+WT MDCK cells cultured in −dox or +dox conditions. (A) Depicted are examples of pre- and post-bleach images of Borg5KD cells (with and without dox) and the corresponding intensity spectrum maps of the teal fluorescent protein (TFP) donor channel at the regions of interest analyzed (white rectangles). (B) n =20 cells/experiment were analyzed for three independent experiments. Error bars show mean±s.d. * P ≤0.05; ns, not significant (unpaired two-tailed t -test). (C,D) Cell clusters after defined trituration of Borg5KD and Borg5KD+WT spheroid cultures in −dox or +dox conditions. Cells were analyzed from 20 random phase contrast images such as those shown in C, in each of three independent experiments. The cumulative frequency distributions are plotted by area for all objects in D. Cumulative distribution as tested by a Kolmogonov–Smirnov test differed between −dox or +dox conditions for Borg5KD with P <0.0001, but not between ±Borg5KD+WT ( P ≈0.1845). (E,F) Phase contrast time lapse (1 frame/min, 30 min) of Borg5KD control (−dox) and Borg5KD (+dox) monolayers that were re-plated at 3×10 4 cells/cm 2 and grown for 24 h. (F) xy -trajectories and total displacement over time was plotted for a group of 7 cells from each monolayer (colored in the stills). Phase contrast images were taken with a 5× (C) or 10× (E) objective.

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): Cdc42EP1/Borg5 (Proteintech, 27904-1-AP, IB 1:500, IF 1:100); Myc tag (clone 9E10, home-made, IB and IF 1:500) or c-Myc tag (Proteintech #16286-1-AP, IF 1:250, IB 1:1000); Vinculin (Novus Biologicals, #NB120-11193, IF 1:100 or Proteintech 26520-1-AP, IF 1:600), ZO1 (clone R26.4C; , 1:50 IF); septin 2 (Proteintech #11397-1-AP, IF 1:300 and #60075-1-Ig, IB 1:1000; BiCell #0022, IF 1:100) Septin 9 (Proteintech # 10769-1-AP, IF 1:300), pThr18/pSer19-MLC2 (Cell Signaling #3674, IF 1:200), E-cadherin (clone RR1, deposited by Barry Gumbiner, University Virginia, with DSHB, Antibody Registry # AB_528114, IF 1:10), tubulin (clone YL1/2, Novus Biologicals, #NB600, IF 1:500), acetylated tubulin clone [6-11B-1] (Millipore Sigma #T6793 IF 1:200), HALO-tag (Promega #g928a, IB 1:500), β-actin [2D4H5] (Proteintech #66009-1-IG, IF: 1:500, IB 1:5000), Actinin α4 (ACTN4; Proteintech 19096-1-AP, IF 1:250), GAPDH [clone1E6D9] (Proteintech # 60004-1-Ig, IB 1:2000), 6xHis-tag [clone 1B7G5] (Proteintech # 66005-1-IG, IB 1:5000), Rac1 (BD Bioscience #610650, IB 1:500), and phospho-tyrosine ([clone PY20], Santa Cruz Biotechnology, sc508, IF 1:200).

Techniques: Cell Culture, Two Tailed Test, Control

Journal: Journal of Cell Science

Article Title: Borg5 restricts contractility and motility in epithelial MDCK cells

doi: 10.1242/jcs.261705

Figure Lengend Snippet: Borg5 depletion and overexpression in mature monolayers alter apical contractility. Borg5KD cells (A–C) or Borg5KD+WT cells (D–F) were first cultured in the absence of dox to a density of 1.5×10 5 cells/cm 2 on Transwell inserts for 24 h and then for an additional 48 h in either −dox (ctrl) or +dox (KD) (A,B,D–F) or cultured, after dox-pre-induction at a plating density of 10 5 cells/cm 2 for 48 h on collagen-coated polyacrylamide hydrogels with an estimated Youngs modulus of 2 kPa (2 kPa PAA) (C). Where indicated, cells were treated for 4 h with 25 µM blebbistatin or DMSO as solvent control. (A,A′) Serial x-y confocal sections from the apical (apex) towards the basal domain of cells labeled for ZO1 and F-actin; note that the F-actin-positive apex rises above the TJs in controls cells, but coincides with TJs in the KD cells (arrowheads). Results are quantified in A′ in 30 cells from 0.3 µm x-y sections as the distance in µm from the most apical point to the plane of their tight junctions; N =2 experiments are presented separately. (B,B′) Overlay of an x-y plane at TJs labeled for ZO1 and at adhesion sites labeled for E-cadherin 2 µm below. (B′) The ratios of individual cell perimeters at TJ to that at the AJ was plotted for 30 cells from N =2 experiments are presented separately. (C,C′) Merged confocal apical and TJ planes and (C′) circularity index at TJs determined from 50 cells in two separate experiments. Indicated are the data set variance coefficients. Violin plots in A′, B′ and C′ highlight median and quartiles with dashed lines. (D) Merged confocal apical and TJ planes showing irregularly sized and shaped surfaces in Borg5KD+WT cells. (E,E′) Views of apical and basal (1 µm above attachment plane) regions of adjacent cells expressing Borg5 at high (blue arrowhead and asterisk) and low (pink arrowhead and asterisk) levels. (E′) Apical to basal surface area ratio in cells from uninduced control cultures and cells in dox-induced cultures separated into Borg5–Myc expressors and non-expressors; data points are collated from N =2 experiments. Error bars shown mean±95% c.i. (F) Apical surface views of depicting Borg5, septin 2 and myosin II (MyoII)-positive F-actin fibers (arrowheads) in irregularly shaped apices. Images in F are representative of two experimental repeats.

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB): Cdc42EP1/Borg5 (Proteintech, 27904-1-AP, IB 1:500, IF 1:100); Myc tag (clone 9E10, home-made, IB and IF 1:500) or c-Myc tag (Proteintech #16286-1-AP, IF 1:250, IB 1:1000); Vinculin (Novus Biologicals, #NB120-11193, IF 1:100 or Proteintech 26520-1-AP, IF 1:600), ZO1 (clone R26.4C; , 1:50 IF); septin 2 (Proteintech #11397-1-AP, IF 1:300 and #60075-1-Ig, IB 1:1000; BiCell #0022, IF 1:100) Septin 9 (Proteintech # 10769-1-AP, IF 1:300), pThr18/pSer19-MLC2 (Cell Signaling #3674, IF 1:200), E-cadherin (clone RR1, deposited by Barry Gumbiner, University Virginia, with DSHB, Antibody Registry # AB_528114, IF 1:10), tubulin (clone YL1/2, Novus Biologicals, #NB600, IF 1:500), acetylated tubulin clone [6-11B-1] (Millipore Sigma #T6793 IF 1:200), HALO-tag (Promega #g928a, IB 1:500), β-actin [2D4H5] (Proteintech #66009-1-IG, IF: 1:500, IB 1:5000), Actinin α4 (ACTN4; Proteintech 19096-1-AP, IF 1:250), GAPDH [clone1E6D9] (Proteintech # 60004-1-Ig, IB 1:2000), 6xHis-tag [clone 1B7G5] (Proteintech # 66005-1-IG, IB 1:5000), Rac1 (BD Bioscience #610650, IB 1:500), and phospho-tyrosine ([clone PY20], Santa Cruz Biotechnology, sc508, IF 1:200).

Techniques: Over Expression, Cell Culture, Solvent, Control, Labeling, Expressing