Journal: The Journal of Cell Biology

Article Title: αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

doi: 10.1083/jcb.200910041

Figure Lengend Snippet: Redistribution of cytosolic and membrane-associated αE-catenin pools affects Arp2/3 complex enrichment in lamellipodia. (A) Representative images from two ActA, β-cat–ActA, and Lyn–β-cat cells fixed and stained with anti-p34 antibody (Arp2/3 complex) and Alexa Fluor–labeled phalloidin (F-actin). Bar, 10 µm. (B) Fluorescence intensity of p34 and F-actin signals in lamellipodia was measured by line scan analysis. Mean fluorescence <3 µm extending from the cell edge (0) in the cell cortex was plotted. 40–50 protrusions from each cell type were measured at three separate points and averaged.

Article Snippet: Arp2/3 was stained using anti–p34-Arc/ARPC2 (1:100; Millipore), and F-actin was stained using Alexa Fluor phalloidin (Invitrogen).

Techniques: Membrane, Staining, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Changes in E-cadherin rigidity sensing regulate cell adhesion

doi: 10.1073/pnas.1618676114

Figure Lengend Snippet: Validation of small molecule inhibitors. (A) MDCK cells adhered to Ecad-Fc substrates and stained for the formin protein FMN1 in the presence or absence of the small molecule inhibitor SMIFH2. (Scale bar, 10 µm.) (B) MDCK cells adhered to Ecad-Fc substrates and stained for the Arp2/3 subunit ARPC2 in the presence or absence of the Arp 2/3 inhibitor CK666. (Scale bar, 10 µm.) (C) GTP-bound Cdc42 isolated from control and ML141-treated cells and analyzed by SDS/PAGE. Bar graph indicates the average from three independent experiments. Activity levels were normalized by setting the control condition to “1.” Error bars represent SEM. (D) GTP-bound Rac isolated from control and NSC23766-treated cells and analyzed by SDS/PAGE. Bar graph indicates the average from three independent experiments. Activity levels were normalized by setting the control condition to 1. Error bars represent SEM.

Article Snippet: The following primary antibodies were used: Arp2 (anti-p34-Arc/ARPC2; Millipore), formin1 (anti-FMN1; Novus Biologicals), and paxillin (anti-paxillin; BD Biosciences).

Techniques: Biomarker Discovery, Staining, Isolation, Control, SDS Page, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Changes in E-cadherin rigidity sensing regulate cell adhesion

doi: 10.1073/pnas.1618676114

Figure Lengend Snippet: Cell adhesion to a 30-kPa and 60-kPa Ecad-Fc PA gel requires the activities of distinct signaling molecules. (A–D) Representative montage of MDCK cells stably expressing E-cadherin:dsRed adhered to a 30-kPa or 60-kPa Ecad-Fc PA gel. Each panel is representative of the effects on cell protrusive area following the addition and washout of different inhibitors: (A) pan-formin inhibitor, SMIFH2; (B) Arp2/3 inhibitor, CK666; (C) Cdc42 inhibitor, ML141; and (D) Rac inhibitor, NSC23766. (Scale bar, 10 µm.) (A–D, Right) Line graphs represent the mean protrusive area for each condition throughout the time course of the experiment. For each line graph, the mean protrusive area of cells on a 30-kPa Ecad-Fc PA gel and on a 60-kPa Ecad-Fc PA gel is indicated by the green and blue lines, respectively. The gray region between the dotted lines indicates the period in which the inhibitor was present. n ≥ 10 cells per condition from at least three independent experiments; error bars represent SEM.

Article Snippet: The following primary antibodies were used: Arp2 (anti-p34-Arc/ARPC2; Millipore), formin1 (anti-FMN1; Novus Biologicals), and paxillin (anti-paxillin; BD Biosciences).

Techniques: Stable Transfection, Expressing

Journal: eLife

Article Title: Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

doi: 10.7554/eLife.42144

Figure Lengend Snippet: ( A ) hiCM allowed to spread for 24 hr in the presence of 25 µM CK666, labeled with actin and α-actinin 2 (i.e., Z-lines) and imaged with SIM. Box indicates presence of MSFs. ( B ) Quantification of percentage of cells with sarcomeres at 24 hr post plating in control and 25 µM CK666. Control: 76 cells, 10 experiments; 25 µM CK666: 41 cells, three experiments. ( C ) Histogram of distribution of distances between α-actinin 2 Z-lines. Note tight distribution of Z-lines in both conditions. Control: 14 cells, three experiments, 317 measurements. 25 µM CK666: 16 cells, three experiments, 530 measurements. ( D ) Stills of hiCM expressing Lifeact-mApple pre (top) and post (bottom) addition of 25 µM CK666 and imaged with spinning disk confocal. Kymographs (right) taken from dotted yellow line (left). ( E ) Rates of retrograde flow of hiCMs depicted as percent change in CK666 from pre-drug condition. 8 cells over three experiments. ( F ) Localization of the Arp2/3 complex in control (top) and 25 µM CK666 treated (bottom) hiCMs imaged with SIM. Note loss of Arp2/3 at the edge of CK666 treated hiCM. Cells spread for 24 hr in presence of 25 µM CK666 as in ( G ) Quantification of loss of the Arp2/3 complex from the leading edge of hiCMs. Control; 36 cells over three experiments. 25 uM CK666; 29 cells over three experiments. ( H ) Live hiCM expressing P16B-mEGFP (a component of the Arp2/3 complex) and imaged with spinning disk confocal. Localization of P16B-mEGFP at leading edge in pre-drug control (top) is acutely lost after addition of 25 µM CK666 (bottom). ( I ) Quantification of hiCMs displaying localization of the Arp2/3 complex (P16B-mEGFP) pre- and post-25µM CK666 in live hiCMs (as in ). 27 cells over three experiments. Scale bars; ( A ) 10 µm low mag, 5 µm high mag inset. ( D ), ( F ), ( H ), 10 µm. P-values denoted in graphs.

Article Snippet: Arp2/3 antibody (Anti-p34-Arc/ARPC2, Millipore Sigma, 07–227) was used at 1:100.

Techniques: Labeling, Expressing

Journal: eLife

Article Title: Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

doi: 10.7554/eLife.42144

Figure Lengend Snippet: ( A ) Actin and myosin II stress fiber formation in non-muscle cells. Actin stress fibers are formed via the Arp2/3 complex and the formin mDia1. NMIIA is the predominant isoform at the leading edge of non-muscle cells, and stress fiber formation is NMIIA dependent. Non-muscle cells display robust retrograde flow of actin stress fibers and display rapid turnover. Large NMIIA stacks are formed via growth and expansion of smaller NMIIA filaments. Citations leading to this model are presented in the cartoon. ( B ) Model of actin and myosin II stress fiber formation in human cardiomyocytes. Sarcomeres are templated by Muscle Stress Fibers (MSFs). MSFs do not require the Arp2/3 complex, and require the formin FHOD3. MSFs display slow retrograde flow compared with non-muscle stress fibers. Both NMIIA and NMIIB are localized to the edge of hiCMs, and display prominent NMII co-filaments. NMIIB-βCMII co-filaments are also present with MSFs. Large βCMII filament stacks form via concatenation and stitching of individual βCMII filaments.

Article Snippet: Arp2/3 antibody (Anti-p34-Arc/ARPC2, Millipore Sigma, 07–227) was used at 1:100.

Techniques:

Journal: eLife

Article Title: Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

doi: 10.7554/eLife.42144

Figure Lengend Snippet:

Article Snippet: Arp2/3 antibody (Anti-p34-Arc/ARPC2, Millipore Sigma, 07–227) was used at 1:100.

Techniques: Derivative Assay, Recombinant, Sequencing, Software