Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Full-length LATS2, LATS2-LATCH and LATS2-LATCH-4mut, were tested for binding to LIMD1 by co-immunoprecipitation. Lysates were prepared from HEK293A cells separately transfected with GFP-tagged LATS2, LATS2-LATCH, LATS2-LATCH-4mut, or LIMD1-V5. Lysates from cells expressing LIMD1-V5 were mixed separately with those expressing the different GFP-LATS2/LATCH constructs, and anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2/LATCH (GFP) and LIMD1 (V5). (B) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH or LATS2-LATCH-4mut. (C) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH treated with (Blebbistatin) or without (DMSO) Blebbistatin.

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Expressing, Construct, Control, Western Blot, Live Cell Imaging, Stable Transfection

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A-B) LIMD1-KO MCF10A cells stably expressing GFP tagged wild-type LIMD1 (WT) or LIMD1 strain insensitive mutants (F512A, F575A, and Y646A, see also .) were established by lentiviral transduction and imaged using fixed and live-cell imaging. (A) The indicated cell lines were stained using anti-LATS1 and anti-TRIP6 antibodies. Merged images show LATS1 (red), TRIP6 (green) and DNA (blue). (B) The indicated cell lines from part (A) were imaged live for GFP fluorescence. (C) Quantification of the LATS1 junction to cytoplasm mean intensity ratio. Error bars represent the standard deviation. ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the plot (mean ± SD; n = 5; ****P < 0.0001). (D-E) LATS1/2 were tested for their ability to bind to strain insensitive mutants of LIMD1 by co-immunoprecipitation. (D) LATS2 was tested for its ability to bind to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1 and LATS2-FLAG were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged WT and mutants of LIMD1 were combined with LATS2-FLAG lysates. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (V5) and LATS2-FLAG (LATS2). (E) LATS1 was tested for binding to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1, and LATS1-3xMyc were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged LIMD1 variants were combined with LATS1-3xMyc lysates. Anti-Myc or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (LIMD1) and LATS1-3xMyc (LATS1). (F) Quantification of relative amounts of LATS2 (normalized to wild-type LIMD1 in the IP fraction) immunoprecipitated by wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants in part (D) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; ***P = 0.001, ****P < 0.0001). (G) Quantification of relative amounts of wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants (normalized to wild-type LIMD1 in the input fraction) immunoprecipitated by LATS1-3xMyc from part (E) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; *P < 0.05, ***P = 0.001).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Stable Transfection, Expressing, Transduction, Live Cell Imaging, Staining, Fluorescence, Standard Deviation, Immunoprecipitation, Transfection, Control, Western Blot, Binding Assay

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS1 or LATS2 N-terminal regions (aa 1-635 in LATS1, aa 1-598 in LATS2) with and without (∆LATCH) the LATCH regions (aa 472-520 in LATS1, aa 418-466 in LATS2) as indicated. (B) AlphaFold2 model showing the three tandem LIM domains of LIMD1 (orange) and two regions of LATS2 (green) that are predicted to interact (LATS-LATCH and the Helical Hairpin). (C) Multiple sequence alignments of LATS1/2 from the indicated species showing the conserved LATCH region. (D-E) The requirement of the conserved LATCH sequence of LATS1/2 to bind with LIMD1 was tested by co-immunoprecipitation. (D) Full-length LATS2 (LATS2) or LATS2 with either the LATCH deleted (LATS2-∆LATCH, aa 418-466 deleted) or with the helical hairpin region deleted (LATS2-∆HH, aa 599-667 deleted) tagged to FLAG and (E) Full-length LATS1 (LATS1) or LATS1 with the LATCH deleted (LATS1-∆LATCH, aa 472-520 deleted) tagged with 3xMyc were tested for binding to LIMD1 by co-immunoprecipitation. FLAG-LATS2 variants, LATS1-3xMyc variants, and LIMD1-V5 were separately transfected in HEK293 cells. HEK293A cell lysates transfected with either FLAG-LATS2 variants (D) or LATS1-3xMyc variants (E) were combined with V5-tagged LIMD1. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2 variants (FLAG) (D) , LATS1 variants (Myc) (E) and LIMD1 (V5).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Sequencing, Immunoprecipitation, Binding Assay, Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: The ability of the LIMD1 and TRIP6 LIM domains to bind to f-actin under strain is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1

doi: 10.1101/2023.10.12.562097

Figure Lengend Snippet: Identification of binding interaction between TRIP6, LATS2, and Vinculin. (A,B) Schematic diagram showing different domains of TRIP6 and LIMD1 (NES: nuclear export signal; LIM: LIM domain; PDZ: PDZ domain binding motif). (C) HEK293 cells were transfected with plasmids expressing vinculin and different FLAG-TRIP6 deletion mutants as indicated. FLAG-TRIP6 immune complexes were isolated from cell lysates and analyzed by western blotting using antibodies against vinculin and FLAG (TRIP6 (FLAG)). Cell lysates (Inputs) were analyzed using antibodies against vinculin and FLAG (TRIP6 (FLAG). Quantification of vinculin levels in TRIP6 immune complexes is shown. (D) HEK293 cells were transfected with plasmids expressing GFP-LATS2 and different FLAG-TRIP6 deletion mutants as indicated. FLAG-TRIP6 immune complexes were isolated from cell lysates and analyzed by western blotting using antibodies against GFP (LATS2 (GFP)) and FLAG (TRIP6 (FLAG)). Cell lysates (Inputs) were analyzed using antibodies against GFP (LATS2 (GFP)) and FLAG (TRIP6 (FLAG)). Quantification of LATS2 levels in TRIP6 immune complexes is shown. (Mean ± SD; n=3; * P≤0.05, *** P≤0.001, T-test). (E) HEK293 cells were transfected with plasmids expressing vinculin and different FLAG-TRIP6 deletion mutants as indicated. FLAG-TRIP6 immune complexes were isolated from cell lysates and analyzed by western blotting using antibodies against vinculin and FLAG (TRIP6 (FLAG)). Cell lysates (Inputs) were analyzed using antibodies against vinculin and FLAG (TRIP6 (FLAG)). Quantification of vinculin levels in TRIP6 immune complexes is shown. (Mean ± SD; n=3; * P≤0.05, *** P≤0.001, T-test). (F) HEK293 cells were transfected with plasmids expressing GFP-LATS2 and different FLAG-TRIP6 deletion mutants as indicated. FLAG-TRIP6 immune complexes were isolated from cell lysates and analyzed by western blotting using antibodies against GFP (LATS2) and FLAG (TRIP6 (FLAG)). Cell lysates (Inputs) were analyzed using antibodies against GFP (LATS2) and FLAG (TRIP6 (FLAG)). (G) Lentiviral infection was used to stably express GFP fusions of wild-type TRIP6 and TRIP6-Δ252-277 in TRIP6 knockout (TRIP6-KO) MCF10A cells. The indicated cell lines were stained using anti-GFP and anti-vinculin antibodies. Merged image shows TRIP6 (green), vinculin (red), and DNA (blue). Scale bar =50microns.

Article Snippet: Antibodies used for immunostaining and western blotting included rabbit anti-GFP (1:500, Cell signaling,2956), mouse anti-Vinculin (1:1000, Sigma, V-9131), mouse anti-TRIP6 (1:1000, Santa Cruz, sc-365122), rabbit anti -LATS1(1:500, Cell Signaling,3477), rabbit anti - LIMD1 (1:500, Novus biologicals, NBP2-56448), rabbit anti α -E-Catenin (1:500, Cell Signaling, 3236).

Techniques: Binding Assay, Transfection, Expressing, Isolation, Western Blot, Infection, Stable Transfection, Knock-Out, Staining

Journal: bioRxiv

Article Title: The ability of the LIMD1 and TRIP6 LIM domains to bind to f-actin under strain is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1

doi: 10.1101/2023.10.12.562097

Figure Lengend Snippet: The TRIP6 and LIMD1 LIM domains are necessary and sufficient for tension dependent localization to adherens junctions. Lentiviral infection was used to stably express GFP fusions of wild-type TRIP6, LIMD1 and various deletion mutants of TRIP6 and LIMD1 in TRIP6 knockout (TRIP6-KO) and LIMD1 knockout (LIMD1-KO) MCF10A cells. For (A) and (B), adherens junction localization of indicated TRIP6 constructs was visualized by co-immunostaining using anti-GFP and anti-Vinculin antibody. For (C) and (D), junction localization of indicated LIMD1 constructs was visualized using GFP fluorescence and immunostaining with anti-TRIP6 antibody. Merged images show TRIP6/LIMD1 (green), Vinculin/TRIP6 (red), and DNA (blue). For (B) and (D), indicated cell lines were treated with DMSO (solvent control) or with blebbistatin before fixation and staining. Scale bar =50microns.

Article Snippet: Antibodies used for immunostaining and western blotting included rabbit anti-GFP (1:500, Cell signaling,2956), mouse anti-Vinculin (1:1000, Sigma, V-9131), mouse anti-TRIP6 (1:1000, Santa Cruz, sc-365122), rabbit anti -LATS1(1:500, Cell Signaling,3477), rabbit anti - LIMD1 (1:500, Novus biologicals, NBP2-56448), rabbit anti α -E-Catenin (1:500, Cell Signaling, 3236).

Techniques: Infection, Stable Transfection, Knock-Out, Construct, Immunostaining, Fluorescence, Solvent, Control, Staining

Journal: bioRxiv

Article Title: The ability of the LIMD1 and TRIP6 LIM domains to bind to f-actin under strain is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1

doi: 10.1101/2023.10.12.562097

Figure Lengend Snippet: Tension dependent localization of TRIP6 and LIMD1 depends on the ability of their LIM domains to bind f-actin under strain. (A-B) TRIP6 and LIMD1 localize to the ends of actin filaments at adherens junctions. F-actin was visualized by staining with fluorescently tagged phalloidin and localization of GFP constructs was visualized by either using anti-GFP antibodies (for TRIP6 GFP constructs) or using GFP fluorescence (for LIMD1 GFP constructs). Top panels (A,B) scale bars =50 microns. Bottom panels (A’,B’) scale bars = 10 microns, represent blow up views of adherens junctions marked by dashed white box in respective top panel. (C-D) Mutations predicted to reduce tension dependent binding to F-actin reduce adherens junction localization of TRIP6 and LIMD1. The indicated cell lines expressing GFP fusions of wild-type or tension sensing mutants of TRIP6 (C) and LIMD1 (D) were stained for GFP for TRIP6 constructs or visualized using GFP fluorescence for LIMD1 constructs and either Vinculin or TRIP6 antibody to mark adherens junctions as indicated. Merged images shows TRIP6/LIMD1(green), Vinculin/TRIP6 (red), and DNA (blue). Scale bars = 50 microns.

Article Snippet: Antibodies used for immunostaining and western blotting included rabbit anti-GFP (1:500, Cell signaling,2956), mouse anti-Vinculin (1:1000, Sigma, V-9131), mouse anti-TRIP6 (1:1000, Santa Cruz, sc-365122), rabbit anti -LATS1(1:500, Cell Signaling,3477), rabbit anti - LIMD1 (1:500, Novus biologicals, NBP2-56448), rabbit anti α -E-Catenin (1:500, Cell Signaling, 3236).

Techniques: Staining, Construct, Fluorescence, Binding Assay, Expressing

Journal: bioRxiv

Article Title: The ability of the LIMD1 and TRIP6 LIM domains to bind to f-actin under strain is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1

doi: 10.1101/2023.10.12.562097

Figure Lengend Snippet: (A) TRIP6 knockout (TRIP6-KO) MCF10A cells stably expressing (via lentiviral transduction) GFP fusions of wild-type TRIP6 (TRIP6) or the indicated tension sensing mutants in each LIM domain were stained for GFP and Vinculin. Merged images show TRIP6 (green), Vinculin (red), and DNA (blue). Scale bars=50microns. (B) Expression of TurboID fusions to indicated TRIP6 constructs was analyzed by western blotting from whole cell lysates of wild-type MCF10A cells (WT) and TRIP6 knockout (TRIP6-KO) MCF10A cells stably expressing the indicated constructs of TRIP6. Blots were probed using antibodies against TRIP6 and GAPDH. (C) Localization of TurboID fusions to the indicated TRIP6 constructs was analyzed by immunostaining of TRIP6 knockout (TRIP6-KO) or wild-type MCF10A cells expressing indicated TurboID tagged TRIP6 constructs or TurboID alone as indicated. Cell lines were stained with antibodies against TRIP6 and fluorescent streptavidin to label biotin (BIOTIN). Merged images shows TRIP6 (green), Biotin (red), and DNA (blue). Scale bars=50microns. (D) Expression levels of LIMD1 in wild type MCF10A cells or LIMD1-KO MCF10A cells stably expressing wild-type (GFP-LIMD1) or the tension sensing mutant (GFP-LIMD1-mut 1,2,3) of LIMD1. Whole cell lysates were probed by Western blotting using antibodies against LIMD1 and GAPDH.

Article Snippet: Antibodies used for immunostaining and western blotting included rabbit anti-GFP (1:500, Cell signaling,2956), mouse anti-Vinculin (1:1000, Sigma, V-9131), mouse anti-TRIP6 (1:1000, Santa Cruz, sc-365122), rabbit anti -LATS1(1:500, Cell Signaling,3477), rabbit anti - LIMD1 (1:500, Novus biologicals, NBP2-56448), rabbit anti α -E-Catenin (1:500, Cell Signaling, 3236).

Techniques: Knock-Out, Stable Transfection, Expressing, Transduction, Staining, Construct, Western Blot, Immunostaining, Mutagenesis

Journal: bioRxiv

Article Title: The ability of the LIMD1 and TRIP6 LIM domains to bind to f-actin under strain is critical for their tension dependent localization to adherens junctions and association with the Hippo pathway kinase LATS1

doi: 10.1101/2023.10.12.562097

Figure Lengend Snippet: Tension dependent association of TRIP6 with LATS1 and LATS1 recruitment to adherens junctions by LIMD1 depends on the ability each protein’s LIM domains to bind f-actin under strain. (A) Protein lysates were prepared from TRIP6 knockout (TRIP6-KO) MCF10A cells expressing TurboID fusions to wild type TRIP6 or indicated TRIP6 constructs along with control wild-type MCF10A cells (WT). Biotinylated protein fractions were pulled down using streptavidin-agarose beads. Protein complexes on beads were analyzed by Western blotting using antibodies against the indicated proteins. Levels of total proteins were analyzed by blotting whole-cell lysates. (B) The indicated cell lines expressing GFP fusions of wild-type or the tension sensing mutant of LIMD1 (LIMD1-mut1,2,3) were imaged for LATS1 immunostaining signal, GFP fluorescence and DNA. Merged images show LIMD1 (green), LATS1 (red), and DNA (blue). Scale bar=50microns.

Article Snippet: Antibodies used for immunostaining and western blotting included rabbit anti-GFP (1:500, Cell signaling,2956), mouse anti-Vinculin (1:1000, Sigma, V-9131), mouse anti-TRIP6 (1:1000, Santa Cruz, sc-365122), rabbit anti -LATS1(1:500, Cell Signaling,3477), rabbit anti - LIMD1 (1:500, Novus biologicals, NBP2-56448), rabbit anti α -E-Catenin (1:500, Cell Signaling, 3236).

Techniques: Knock-Out, Expressing, Construct, Control, Western Blot, Mutagenesis, Immunostaining, Fluorescence