Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a , b ) Distribution of myosin-IIa and -IIb in control and EpCAM-depleted cells. Scale bars, 5 μm. ( c , d ) Distribution of E-cadherin and myosin-IIa, or E-cadherin and myosin-IIb in EpCAM-depleted cells. Scale bars, 5 μm. ( e , f ) 3D-SIM analysis of E-cadherin and myosin-IIb, or Crb3 and myosin-IIa in EpCAM-deprived cells. Scale bars, 5 μm. ( g ) Western blot analysis of MLC2 and P-MLC2 amounts in control and EpCAM-depleted cells. GAPDH was used as loading control. ( h ) Quantification of P-MLC2 amount relative to MLC2 in control and EpCAM-depleted cells. One-way ANOVA with Dunnett's test, * P <0.01. Caco2shNT=1, Caco2shEpCAM#1=0.11±0.2, Caco2shEpCAM#2=0.42±0.2. ( i ) Distribution of P-MLC2 in control or EpCAM-silenced cells. Scale bars, 5 μm. ( j ) Quantification of total P-MLC2 immunofluorescence intensity relative to MLC2 immunofluorescence intensity in control or EpCAM-depleted cells. One-way analysis of variance test with Dunnett's test, * P =0.007, ** P =0.002. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =30, n (Caco2shEpCAM#2) =30. Caco2shNT=1, Caco2shEpCAM#1=0.4±0.05, Caco2shEpCAM#2=0.52±0.07. ( k ) Quantification of P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at bicellular junctions (BJs) in control or EpCAM-depleted cells. One-way analysis of variance with unpaired t -test, * P =0.0001. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =28, n (Caco2shEpCAM#2) =30. Caco2shNT=1, Caco2shEpCAM#1=6.01±1.24, Caco2shEpCAM#2=4.1±0.33. ( l – n ) Distribution of myosin-IIa, -IIb and P-MLC2 in control or CTE biopsies. Scale bars, 5 μm. N (Control) =3 biopsies, N (CTE) =3. ( o – q ) Quantification of myosin-IIa, -IIb or P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at BJs in control or CTE biopsies. N (Control) =3 biopsies, N (CTE) =3. Unpaired t -test, ( o ) * P <0.0001, ( p ) * P =0.0062, ( q ) * P =0.0016. n (Control myosin-IIa) =37 cells, n (CTE myosin-IIa) =29, n (Control myosin-IIb) =14, n (CTE myosin-IIb) =14, n (Control P-MLC2) =15, n (CTE P-MLC2) =21. Myosin-IIa (Control) =1±0.04, myosin-IIa (CTE) =5.86±0.54, myosin-IIb (Control) =1±0.08, myosin-IIb (CTE) =2.27±0.39, P-MLC2 (Control) =1±0.07, P-MLC2 (CTE) =3.25±0.62. For quantification, three independent replicates have been performed. Values are mean±s.e.m. Nuclei were stained with Hoechst 33342.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Control, Western Blot, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a ) Quantification of P-MLC2 immunofluorescence intensity at TCs relative to immunofluorescence intensity at BJs in control, EpCAM-depleted or 2h-blebbistatin-treated EpCAM-depleted cells. One-way analysis of variance with unpaired t -test, * P <0.05. n (Caco2shNT) =30 cells, n (Caco2shEpCAM#1) =28, n (Caco2shEpCAM#2) =30, n (Caco2shEpCAM#1+Blebbistatin 50 μM) =31, n (Caco2shEpCAM#2+Blebbistatin 50 μM) =30. Caco2shNT=1, Caco2shEpCAM#1=6.01±1.24, Caco2shEpCAM#2=4.1±0.33, Caco2shEpCAM#1+Blebbistatin 50 μM=1.57±0.17, Caco2shEpCAM#2+Blebbistatin50 μM=0.91±0.06. ( b ) SEM analyses of apical surfaces of DMSO-treated control, EpCAM-depleted or EpCAM-depleted cells treated with blebbistatin for 2 h. Scale bars, 4 μm. ( c ) Confocal analysis and 3D rendering of z -stacks of actin location at TCs in DMSO-treated control, EpCAM-depleted or EpCAM-depleted cells treated with blebbistatin for 2 h. Scale bars, 4 μm. ( d ) High magnifications of EADs from SEM or confocal and 3D rendering analyses. Scale bars, 1 μm. ( e ) Quantification of EADs on DMSO or blebbistatin treatment in Caco2shNT or shEpCAM cells. One-way ANOVA with Tukey's test, **** P <0.0001. n (Caco2shNT+DMSO) =107 cells, n (Cao2shEpCAM#1+DMSO) =105, n (Caco2shEpCAM#2+DMSO) =91, n (Caco2shEpCAM#1+Blebbistatin) =96, n (Caco2shEpCAM#2+Blebbistatin) =81. EAD-positive Caco2shNT cells=0%, EAD-positive Caco2shEpCAM#1+DMSO cells=87.41±1.64%, EAD-positive Caco2shEpCAM#1+DMSO cells=91.98±4.21%, EAD-positive Caco2shEpCAM#1+blebbistatin cells=9.55±5.46%, EAD-positive Caco2shEpCAM#1+DMSO=18.43±1.83%. ( f ) Confocal analysis of actin distribution and brush border formation in Caco2shEpCAM cells. Cells were treated for 2 h with DMSO, or for 10 min or 2 h with 5 or 50 μM of blebbistatin. Scale bars, 5 μm. ( g ) Quantification of EADs on DMSO or blebbistatin treatment in Caco2 shEpCAM cells. N (2 h, DMSO)=5,467 cells, N (10 min, 5 μΜ)=3,598, N (10 min, 50 μΜ)=4,032, N (2 h, 5 μΜ)=289, N (2 h, 50 μΜ)=4,416. t -test, P <0.0001. ( h ) Analysis of lateral membrane behaviour in EpCAM-deprived Caco2 cells during 50 μM blebbistatin treatment and time-lapse acquisitions. Lateral membranes have been stained with SirActin labelling. Scale bars, 10 μm. For quantification, three independent replicates have been performed. Values are mean±s.e.m. Nuclei were stained with Hoechst 33342.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Immunofluorescence, Control, Membrane, Staining

Journal: Nature Communications

Article Title: Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

doi: 10.1038/ncomms13998

Figure Lengend Snippet: ( a ) Statistical analysis of the number of tuft-like structures detected along the 3D microfabricated villus structures in control ( Caco2 shNT ), EpCAM-depleted cells ( Caco2 shEpCAM ) and 2h- blebbistatin-treated EpCAM-depleted cells ( Caco2 shEpCAM#1+Blebbistatin 50 μ M ). Three independent replicates have been performed. One-way analysis of variance with unpaired t -test, * P <0.0001. n (Caco2 shNT) =196 villi, n (Caco2 shEpCAM) =210 villi, n (Caco2 shEpCAM+Blebistatin) =210 villi. Caco2 shNT(mean number of tufts per villus)=5, Caco2 shEpCAM(mean number of tufts per villus)=12, Caco2 shEpCAM+Blebbistatin 50 μM(mean number of tufts per villus)=6. ( b – d ) Confocal microscopy analysis of myosin-IIa ( b ), myosin-IIb ( c ) and P-MLC2 ( d ) distribution in control ( Caco2 shNT ) and EpCAM-silenced ( Caco2 shEpCAM ) cells that were grown on villous PDMS inserts or in 3D Matrigel cultures for 21 days. Transversal xy views are presented. Scale bars, 50 μm. ( e ) Schemes recapitulating the cellular and epithelial phenotypes observed in control ( Caco2 shNT ) or EpCAM-depleted ( Caco2 shEpCAM ) conditions when cells are grown in 2D, 3D synthetic villi or 3D Matrigel cultures. Contractile apparatus (blue) and predictated tensile forces (red arrows) are presented.

Article Snippet: Rabbit polyclonal antibody directed against P-MLC2 (S19/S20, #600-401-416, IF dilution 1:100, WB dilution 1:500) was from Rockland (Limerick, PA, USA).

Techniques: Control, Confocal Microscopy

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection

doi: 10.3389/fcimb.2018.00415

Figure Lengend Snippet: Recruitment of MYPT1 and Myosin phosphatase pathway components and extrusion production by C. trachomatis L2-wild type and L2-ΔCT228. HeLa cell monolayers were infected at a MOI of ~0.5 with L2-wild type and L2-ΔCT228 for 18 h (in technical triplicate). Cells were fixed and stained with primary antibodies to MYPT1, Chlamydia LPS, MLC2 (pS19), Src Y474, MLCK (pY471), non-muscle Myosin IIa and IIb followed by fluorescent secondary antibodies. Experiments were repeated on three separate occasions and representative images were selected. (A,B) Top panel shows individual and merged images of MYPT1 recruitment (green) and Chlamydia LPS staining (red) in both the L2-wild type and L2-ΔCT228. Lower panel of individual and merged images show MLC2 (pS19), MLCK (pY471), and Mysoin IIa and IIb (green) co-localizing with active Src Y474 kinase (red) in microdomains at the periphery of inclusions in both L2-wild type and L2-ΔCT228. Scale bar, 10 μm. (C) Total protein from L2-wild type and L2-ΔCT228 infected HeLa cells at 24 and 48 h post-infection were assessed for MLC2, MLC2 (pS19), HsP60, and GAPDH levels by western blot analysis. (D) HeLa cells were treated with either Scramble (Scr) or MYPT1 siRNA for 48 h prior to infection with L2-wild type and L2-ΔCT228. Protein samples were assessed for MYPT1 and GAPDH levels by western blot. (E) Extrusions collected and (F) IFUs were assessed for L2 wild-type and L2-ΔCT228 at 48 h post-infection in either Scramble (symbols and solid bars) or MYPT1 (open symbols and white bars) siRNA treated HeLa cells. * p < 0.0001.

Article Snippet: Primary antibodies to MLC2 (Millipore Sigma, Burlington, MA), p-MLC2 (S19) (ThermoFisher Scientific), HsP60 (ThermoFisher Scientific), GAPDH (Santa Cruz), and MYTP1 (Cell Signaling, Danvers, MA) were used herein.

Techniques: Infection, Staining, Western Blot