Journal: bioRxiv

Article Title: MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

doi: 10.1101/409110

Figure Lengend Snippet: ( A ) Immunofluorescent staining of different Golgi marker, the matrix proteins GM130 (green), the cis -Golgi protein Giantin (red) and the trans -Golgi network protein TGN46 (magenta) in representative control (top) and MYO1C depleted (bottom) hTertRPE-1 cells in classical, unconstrained culture conditions. The nucleus is stained with Dapi (blue). Quantification of the 2D projected Golgi area in control conditions (n=145) and upon MYO1C depletion (n=165). Error bars represent the standard deviation of three independent experiments. *** indicates P-value < 1×10 −4 in a Student T-test. ( C ) Fluorescent images of representative hTertRPE-1 cells expressing GFP or GFP-MYO1C (green) and stained for the Golgi apparatus (red, GM130). ( D ) Quantification of the 2D projected Golgi area (stained by GM130) of non transfected cells and cells as in C. Error bars represent the standard deviation of three independent experiments with n > 40 cells of each condition. * indicates P-value < 1×10 −2 in a Student T-test on averages of three independent experiments ( E ) Electron microscopy images of representative intracellular areas of unconstrained hTertRPE-1 cells containing the Golgi apparatus in control conditions and upon MYO1C depletion. Scale bar: 1 µ m.

Article Snippet: We used the following mouse monoclonal antibodies α-GM130 (BD Biosciences cat: 610823), α-MYO1C (Santa Cruz sc-136544) and α-actin (Sigma, A-2228), rabbit polyclonal antibodies α-Lamp1 (Sigma, L1418 or Abcam, Ab24170), human monoclonal antibody (F2C-hFc) α-tubulin and α-Giantin (Recombinant Protein and Antibody Platform of the Institut Curie) and sheep polyclonal antibody α-TGN46 (Bio-Rad, AHP500).

Techniques: Staining, Marker, Standard Deviation, Expressing, Transfection, Electron Microscopy

Journal: bioRxiv

Article Title: MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

doi: 10.1101/409110

Figure Lengend Snippet: ( A ) Fluorescent images of a representative, unconstrained hTertRPE-1 cell stained with MYO1C antibody to visualize the endogenous protein (green), TGN46 antibody to visualize the Golgi apparatus (red) and Dapi to visualize the nucleus (blue). ( B, C ) Fluorescent images of representative, micropatterned hTertRPE-1 cells expressing GFP-MYO1C (green) and stained for the Golgi apparatus either with TGN46 or Giantin antibody (red) and the nucleus with Dapi (blue). ( D ) Fluorescent images of a time-lapse acquisition of unconstrained hTertRPE-1 cells expressing GFP-MYO1C (green) and mCherry-Rab6 (red). Scale bar: 10 µm.

Article Snippet: We used the following mouse monoclonal antibodies α-GM130 (BD Biosciences cat: 610823), α-MYO1C (Santa Cruz sc-136544) and α-actin (Sigma, A-2228), rabbit polyclonal antibodies α-Lamp1 (Sigma, L1418 or Abcam, Ab24170), human monoclonal antibody (F2C-hFc) α-tubulin and α-Giantin (Recombinant Protein and Antibody Platform of the Institut Curie) and sheep polyclonal antibody α-TGN46 (Bio-Rad, AHP500).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

doi: 10.1101/409110

Figure Lengend Snippet: ( A ) Fluorescent images of a representative, unconstrained hTertRPE-1 cell stained with MYO1C antibody to visualize the endogenous protein (green), TGN46 antibody to visualize the Golgi apparatus (magenta) and phalloidin to visualize the actin cytoskeleton (red). The nucleus is stained with Dapi (blue). ( B ) Fluorescent images of representative, micropatterned hTertRPE-1 cells expressing GFP-MYO1C (green) and stained with phalloidin to visualize F-actin (red) and Dapi to visualize the nucleus (blue). ( C ) Domain structure of MYO1C, the mutant form MYO1CΔABL and the truncated Tail domain. ( D ) Fluorescent images of representative hTertRPE-1 cells expressing GFP-MYO1C, GFP-MYO1CΔABL or GFP-Tail (green), stained for the Golgi apparatus (magenta, GM130) and F-actin with phalloidin (red). ( E ) Quantification of GFP-positive spots at the Golgi area as in D for GFP (n=24), GFP-MYO1C (n=49), GFP-MYO1CΔABL (n=56) or GFP-Tail (n=34). % of cells is shown that contain 0 (light grey), 1-10 (dark grey) or more than 10 (black) Golgi-associated spots per cell. Note that control (GFP-expressing) cells never contained more than 1 Golgi-associated spot per cell.

Article Snippet: We used the following mouse monoclonal antibodies α-GM130 (BD Biosciences cat: 610823), α-MYO1C (Santa Cruz sc-136544) and α-actin (Sigma, A-2228), rabbit polyclonal antibodies α-Lamp1 (Sigma, L1418 or Abcam, Ab24170), human monoclonal antibody (F2C-hFc) α-tubulin and α-Giantin (Recombinant Protein and Antibody Platform of the Institut Curie) and sheep polyclonal antibody α-TGN46 (Bio-Rad, AHP500).

Techniques: Staining, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

doi: 10.1101/409110

Figure Lengend Snippet: ( A ) Western blot analysis of actin and GAPDH expression levels in control and MYO1C depleted hTertRPE-1 cells (top) and densitometry quantification (bottom). Error bars represent the standard deviation of three independent experiments. ( B ) Fluorescent images representing the maximal intensity projections (MIP) of F-actin stained by phalloidin in representative control and MYO1C depleted hTertRPE-1 cells seeded on a crossbow-shaped micropattern (top). Average intensity projection of the MIP images from n cells from three independent experiments. Scale bar: 10 µm. ( C ) Fluorescent images of a representative, unconstrained hTertRPE-1 cells stably expressing GFP-Rab6 in control conditions and upon treatment with Latrunculin A and CK666. ( D ) Quantification of the 2D projected Golgi area (visualized by GFP-Rab6) in control conditions (n=14) and upon treatment with Latrunculin A (LatA, n=19) and CK666 (n=15). Error bars represent the standard deviation of individual cells. * indicates P-value < 1×10 −2 in a Student T-test. ( E ) Fluorescent images representing the maximal intensity projections (MIP) of the Golgi apparatus (visualized by GFP-Rab6) in single, representative hTertRPE-1 cells in control conditions and ACTR3 depletion on a crossbow-shaped micropattern. Scale bar: 10 µm. ( F ) Quantification of the 2D projected Golgi area (visualized by GFP-Rab6) in control conditions (n=25) and ACTR3 depleted hTertRPE-1 cells (n=26). Error bars represent the standard deviation of individual cells. *** indicates P-value < 1×10 −4 in a Student T-test.

Article Snippet: We used the following mouse monoclonal antibodies α-GM130 (BD Biosciences cat: 610823), α-MYO1C (Santa Cruz sc-136544) and α-actin (Sigma, A-2228), rabbit polyclonal antibodies α-Lamp1 (Sigma, L1418 or Abcam, Ab24170), human monoclonal antibody (F2C-hFc) α-tubulin and α-Giantin (Recombinant Protein and Antibody Platform of the Institut Curie) and sheep polyclonal antibody α-TGN46 (Bio-Rad, AHP500).

Techniques: Western Blot, Expressing, Standard Deviation, Staining, Stable Transfection

Journal: bioRxiv

Article Title: MYO1C facilitates arrival at the Golgi apparatus through stabilization of branched actin

doi: 10.1101/409110

Figure Lengend Snippet: We propose that MYO1C accumulates at the proximity of the Golgi apparatus, on non-Golgi membranes, by its PH domain and stabilizes branched actin at the Golgi that facilitates arrival of incoming transport carriers at the Golgi apparatus.

Article Snippet: We used the following mouse monoclonal antibodies α-GM130 (BD Biosciences cat: 610823), α-MYO1C (Santa Cruz sc-136544) and α-actin (Sigma, A-2228), rabbit polyclonal antibodies α-Lamp1 (Sigma, L1418 or Abcam, Ab24170), human monoclonal antibody (F2C-hFc) α-tubulin and α-Giantin (Recombinant Protein and Antibody Platform of the Institut Curie) and sheep polyclonal antibody α-TGN46 (Bio-Rad, AHP500).

Techniques: