Journal: The Journal of cell biology

Article Title: An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells.

doi: 10.1083/jcb.201704085

Figure Lengend Snippet: Figure 2. The apicosome has features of the cellular exterior. (A and B) TEM images of single H9 cells grown under feeder-free conditions on a Geltrex- coated Thermanox substrate (A) or on a tissue culture plate (B) for 20 h. (B-i) Magnified image of the boxed region in B, showing a primary cilium within the apicosome. (C) Fluorescent confocal image of a single H9 cell stained with ciliary (ARL13B) and centrosomal (γ-TUBULIN) markers as well as HOECHST (blue). (D) H9 cell expressing EZRIN–EGFP (top), stained with ER-Tracker (red); isolated H9 cell stained with Fluo-3-AM (bottom, green) and ER-Tracker (red). (E) H9 cells expressing m-TdTomato (top) or Lifeact–mCherry (bottom), stained with Fluo-3-AM. (F) Schematic of an isolated hPSC with an internal apicosome, containing microvilli (green protrusions), a primary cilium (red), and accumulated Ca2+. Bars: (A and B) 5 µm; (B-i) 500 nm; (C and E) 10 µm.

Article Snippet: The piggyBac transposon system and human MDIA1-GFP constructs were generously provided by Drs. Joseph LoTurco and Shuh Narumiya, respectively. pQC membrane TdTomato was a gift from Connie Cepko (plasmid 37351; Addgene). pTK_93-Lifeact–mCherry was a gift from Iain Cheeseman (plasmid 46357; Addgene).

Techniques: Staining, Expressing, Isolation

Journal: Journal of the American Society of Nephrology

Article Title: Inverted Formin 2 Regulates Actin Dynamics by Antagonizing Rho/Diaphanous-related Formin Signaling

doi: 10.1681/asn.2012080834

Figure Lengend Snippet: Figure 1. INF2 maintains podocyte cortical actin by opposing Rho/mDia activity. (A) Podocytes (undifferentiated) were respectively transfected with universal negative control siRNA duplex (control), INF2-targeting siRNA (INF2 KD), INF2 KD+DN-RhoA, INF2 KD+DN- mDia1, or INF2 KD, followed by treatment with cell permeable C3 transferase (Cytoskeleton Inc.; Rho inhibitor I, 1.0 mg/ml for 2 hours). Cortactin, DN RhoA (DN mDia1), and actin filaments (F-actin) were co-illustrated by immunofluorescent stain (Cortactin in red, DN- RhoA/DN mDia1 in green, and F-actin in blue). (B) INF2 knockdown by siRNA was confirmed by Western blotting. (C) High transfection efficiency of siRNA as reflected by Cy3-labeled siRNA duplex. The transfection efficiency was calculated as the number of Cy3-positive cells/100 cells with positive stain of DAPI. Both the relative area of cortical actin mesh (D) and the relative length of cortactin-positive membrane (E) were calculated for 20 randomly selected cells in each group; data were expressed as mean 6 standard deviation. The difference among groups was statistically analyzed using one-way ANOVA and least significant difference test. *P,0.05 versus cells with INF2 KD only.

Article Snippet: Human nephrin was cloned into pcDNA3.1 by PCR (non- tagged). pEGFP-mDia1DN3 (HindIII) was a generous gift from Dr. Shuh Narumiya, Department of Pharmacology, Kyoto University, Japan.44 RNA Interference Human podocytes were transfected with INF2-targeting Dicer- Substrate siRNA duplexes (TriFECTa kit from IDT) using Hyperfect transfection reagent (Qiagen) at a final concentration of 10 nM.

Techniques: Activity Assay, Transfection, Negative Control, Control, Staining, Knockdown, Western Blot, Labeling, Membrane, Standard Deviation

Journal: PLoS ONE

Article Title: ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

doi: 10.1371/journal.pone.0055211

Figure Lengend Snippet: Chemotaxis of T47D cells expressing dominant negative (DN) RhoA, Rac1 or Cdc42 or a control vector (Ctrl) in response to a HRG gradient was analyzed as described in . ( A ) Cell velocity was calculated from individual tracks; mean+/−s.e.m. is shown; * p<0.01. ( B ) Rose diagrams and Rayleigh tests; p>0.05 is considered uniform distribution. Cdc42 and RhoA are involved in oriented migration. ( C ) Implication of the Memo/GSK3/ACF7 signaling pathway in chemotaxis. Directional distribution (upper panels) of cells expressing control (Ctrl), Memo or ACF7 siRNA, or active GSK3 or membrane targeted ACF7 (ACF7-CCKVL) constructs was evaluated. (Lower panels) Still images of EGFP-tubulin expressing cells, analyzed by time-lapse fluorescence microscopy 30 min after addition of HRG, are shown. Insets: zooms showing the presence or absence of peripheral microtubules in cell protrusions.

Article Snippet: When indicated, cells were also transfected with the following cDNA constructs: EGFP-α-tubulin (Clontech, Saint-Germain-en-Laye, France), Flag-mDia1ΔN3 (S. Narumiya, Kyoto University, Japan), APC (B. Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore; Addgene plasmid 16507), GSK3β S9A, GSK3β K85A (J. Woodgett, Mount Sinai Hospital, Toronto; Addgene plasmids 14754/14755) and EGFP-ACF7-NC (E. Fuchs, Rockefeller University, New York).

Techniques: Chemotaxis Assay, Expressing, Dominant Negative Mutation, Control, Plasmid Preparation, Migration, Membrane, Construct, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

doi: 10.1371/journal.pone.0055211

Figure Lengend Snippet: Velocity of SKBr3 cells transfected with control or PLCγ siRNA in response to a HRG gradient was assayed as described in . ( A ) Cell velocity was calculated from individual tracks; mean+/−s.e.m. is shown; * p<0.01. ( B ) Rose diagrams and Rayleigh tests; p>0.05 is considered random migration. ( C ) Schematic of the Memo/ACF7 pathway. ( D ) EGFP-tubulin expressing cells were visualized by time-lapse microscopy (see illustrations in supplementary material ) and the percentage of cells with peripheral microtubules (MTs) was evaluated 30 min after addition of HRG. Cells were transfected with Memo, PLCγ or control (Ctrl) siRNA, together with active mDia1, dominant negative GSK3 (DN-GSK3), ACF7 minigene (ACF7) or membrane targeted ACF7 minigene (ACF7-CCKVL). 90–150 cells were counted per condition in three independent experiments; mean+/−s.e.m. is shown; * p<0.01. Expression of DN-GSK3 or membrane targeted ACF7 rescued microtubules in both Memo and PLCγ-depleted cells, while active mDia1 restored microtubules only in Memo-depleted cells.

Article Snippet: When indicated, cells were also transfected with the following cDNA constructs: EGFP-α-tubulin (Clontech, Saint-Germain-en-Laye, France), Flag-mDia1ΔN3 (S. Narumiya, Kyoto University, Japan), APC (B. Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore; Addgene plasmid 16507), GSK3β S9A, GSK3β K85A (J. Woodgett, Mount Sinai Hospital, Toronto; Addgene plasmids 14754/14755) and EGFP-ACF7-NC (E. Fuchs, Rockefeller University, New York).

Techniques: Transfection, Control, Migration, Expressing, Time-lapse Microscopy, Dominant Negative Mutation, Membrane

Journal: PLoS ONE

Article Title: ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

doi: 10.1371/journal.pone.0055211

Figure Lengend Snippet: ( A ) SKBr3 cells were transfected with EGFP-tubulin and Stathmin (Stath) siRNA, and treated with LY294004 (PI3K inhib) or LiCl to inhibit GSK3 activity, as indicated, before addition of HRG. The percentage of cells with captured or disassembled microtubules was evaluated; * p<0.01 relative to the respective controls. ( B ) Cells loaded on a Dunn chamber were treated as in A, for analysis of the chemotactic response. Rose diagrams and Rayleigh tests are shown. p>0.05 is considered random migration. ( C ) T47D cells were pre-treated with the indicated inhibitor before addition of HRG. Phosphorylation of Stathmin was evaluated by Western blotting using an antibody against phosphorylated Ser16. An anti-tubulin antibody was used to assess protein loading. Phosphorylation of Stathmin is dependent on PI3K activity. ( D ) Cells were transfected with control or Stathmin siRNA, before treatment with the indicated inhibitor. After treatment with HRG, cells were fractionated and the microtubule-enriched fraction was analyzed by Western blotting using an anti-tubulin antibody. Cell lysates were also probed for Stathmin and RhoGDI (loading control). Depletion of Stathmin prevents microtubule disassembly induced by the Akt inhibitor. ( E ) Cells were transfected with DN-Rac1, DN-Cdc42 or pre-treated with the Akt inhibitor, before addition of HRG and analysis of phosphorylated PAK1, PAK1, GFP (for expression of DN-Cdc42 and DN-Rac1) and RhoGDI (loading control). Phosphorylation of PAK1 is dependent on Akt signaling. ( F ) Cells were transfected with DN-PAK1 or pre-treated with a PAK or Akt inhibitor, before treatment with HRG for 30 min and analysis of phosphorylated Stathmin, Stathmin and PAK1 by Western blotting. ( G ) Cells were transfected with EGFP-tubulin, active GSK3 and/or DN-PAK1. The percentage of cells with captured or disassembled microtubules was evaluated.

Article Snippet: When indicated, cells were also transfected with the following cDNA constructs: EGFP-α-tubulin (Clontech, Saint-Germain-en-Laye, France), Flag-mDia1ΔN3 (S. Narumiya, Kyoto University, Japan), APC (B. Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore; Addgene plasmid 16507), GSK3β S9A, GSK3β K85A (J. Woodgett, Mount Sinai Hospital, Toronto; Addgene plasmids 14754/14755) and EGFP-ACF7-NC (E. Fuchs, Rockefeller University, New York).

Techniques: Transfection, Inhibition, Activity Assay, Migration, Phospho-proteomics, Western Blot, Control, Expressing

Journal: PLoS ONE

Article Title: ErbB2-Dependent Chemotaxis Requires Microtubule Capture and Stabilization Coordinated by Distinct Signaling Pathways

doi: 10.1371/journal.pone.0055211

Figure Lengend Snippet: Our data show that ErbB2 controls microtubules via distinct, but complementary, signaling pathways. The Memo/RhoA and the PLCγ/cPKC pathways converge to control ACF7 localization at the leading edge, allowing microtubule capture. While PKCζ is also required downstream of PLCγ, its mode of action is still unknown. The PI3K/Akt pathway contributes to microtubule stability, at later stages, and involves the combined effect of the PAK/Stathmin pathway and repression of GSK3 activity. Microtubule capture and stabilization allow the chemotactic response to HRG.

Article Snippet: When indicated, cells were also transfected with the following cDNA constructs: EGFP-α-tubulin (Clontech, Saint-Germain-en-Laye, France), Flag-mDia1ΔN3 (S. Narumiya, Kyoto University, Japan), APC (B. Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore; Addgene plasmid 16507), GSK3β S9A, GSK3β K85A (J. Woodgett, Mount Sinai Hospital, Toronto; Addgene plasmids 14754/14755) and EGFP-ACF7-NC (E. Fuchs, Rockefeller University, New York).

Techniques: Protein-Protein interactions, Control, Activity Assay