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

Journal: Developmental cell

Article Title: LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to force.

doi: 10.1016/j.devcel.2021.04.002

Figure Lengend Snippet: Figure 3. LIMD1 phase separation is regulated by phosphorylation on its IDR (A) Representative images of LIMD1 KO MDA-MB-231 cells expressing Opto-LIMD1, Opto-DLIM, or Opto-DIDR upon blue light exposure. Scale bar, 20 mm. (B) Phase diagrams of DLIM and DIDR in different protein and NaCl concentration. The red circles indicate phase separation, and the blue circles indicate no phase separation. (C) Schematic representation of 9 phosphorylation sites on the IDR of LIMD1. Table summary of the 9 phosphorylation sites on IDR, which were mutated to aspartic acid (D) or alanine (A), respectively, to generated +Phos or Phos mutants. (D) Quantification of droplets number upon blue light exposure. LIMD1 KO MDA-MB-231 cells were transfected with Opto-LIMD1 (WT), Opto-LIMD1 phos- phorylation-mimetic mutant (+Phos), and Opto-LIMD1 phosphorylation deficient mutant (Phos), respectively, and treated with 5 mM Ro-3306 (CDK1 inhibitor). Cells with similar fluorescent intensity before illumination were selected for the analysis (NWT = 5 cells, N+Phos = 7 cells, NPhos = 7 cells, error bar: mean with SEM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-GAPDH Abcam Cat# ab181602; RRID: AB_2630358 Rabbit polyclonal anti-GFP MBL International Cat# 598; RRID: N/A Mouse monoclonal anti-GFP MBL International Cat# M048-3; RRID: AB_591823 Mouse monoclonal anti-DDDDK-tag MBL International Cat# M185-3L; RRID: AB_11123930 Mouse monoclonal anti-Paxillin BD Biosciences Cat# 610620; RRID: AB_397952 Rabbit polyclonal anti-LIMD1 Thermo Fisher Scientific Cat# PA5-28491; RRID: AB_2545967 Mouse monoclonal anti-LIMD1 EMD_Millipore Cat# MABD85; RRID: N/A Alexa Fluor 488 Thermo Fisher Scientific Cat# A-21202; RRID: AB_141607 Alexa Fluor 555 Thermo Fisher Scientific Cat# A-31570; RRID: AB_2536180 Bacterial and virus strains E. coli BL21 (DE3) Homemade N/A Chemicals, peptides, and recombinant proteins Myosin II inhibitor (blebbistatin) EMD_Millipore Cat# 2946047 Rho activator II Cytoskeleton Cat# CN03 ROCK Inhibitor (Y-27632) Cell Signaling Technology Cat# 13624S CDK1 inhibitor (Ro-3306) Selleck Cat# S7747 phosphatase inhibitor cocktail 3 Sigma-Aldrich Cat# P0044 protease inhibitor mini tablets, EDTA-free Thermo Fisher Scientific Cat# 88666 FluoSpheres carboxylate-modified beads conjugated with 561 dye (0.1 mm) Thermo Fisher Scientific Cat# F8887 Protein A+G agarose beads Beyotime Cat# P2012 SF-900TM II SFM GIBCO Cat# 10902088 SIM SF Medium Sino Biological Inc. Cat# MSF1 Fibronectin human plasma Sigma-Aldrich Cat# F1056-5MG EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl) Thermo Fisher Scientific Cat# 22980 MES (2-(N-Morpholino) ethanesulfonic acid, 4-Morpholineethanesulfonic acid) Sigma-Aldrich Cat# M3671-50G NHS (N-Hydroxysuccinimide) Thermo Fisher Scientific Cat # 24500 Critical commercial assays Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit Transgene Cat# AT311-02 Chelating Beads 6FF Smart Life Sciences Cat# SA051500 Superdex 200 Increase, 10/300 GL GE Healthcare Cat# 28-9909-44 Superose 6 Increase, 10/300 GL GE Healthcare Cat# 29-0915-96 Dextrin Beads Smart Lifesciences Cat# SA077010 Experimental models: cell lines Mouse: MEF cells Provided from James Bear at University of North Carolina at Chapel Hill, USA N/A Human: HEK 293T cells Provided from Yuxin Yin at Peking University, China N/A Human: MDA-MB-231 cells Provided from Yujie Sun at Peking University, China N/A Spodoptera frugiperda 9 (Sf9) Invitrogen Cat# 11496015 (Continued on next page) e1 Developmental Cell 56, 1313–1325.e1–e7, May 3, 2021

Techniques: Phospho-proteomics, Expressing, Concentration Assay, Generated, Transfection, Mutagenesis