Open Access Highly Accessed Research article

R-Ras regulates β1-integrin trafficking via effects on membrane ruffling and endocytosis

Matthew W Conklin1, Aude Ada-Nguema1, Maddy Parsons2, Kristin M Riching1 and Patricia J Keely1*

Author Affiliations

1 Dept of Pharmacology, Laboratory for Molecular Biology and the University of Wisconsin Carbone Cancer Center, University of Wisconsin, 1525 Linden Dr, Madison, WI, 53706, USA

2 Randall Division of Cell and Molecular Biophysics, King's College London Guy's Campus, London, SE1 1UL, UK

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BMC Cell Biology 2010, 11:14  doi:10.1186/1471-2121-11-14

Published: 18 February 2010

Additional files

Additional file 1:

GFP-alone control. Representative cell that was transfected with GFP alone showed no localization of the fluorophore to membranes.

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Additional file 2:

Movie of GFP-R-Ras(wt) dynamics. Timelapse images of GFP-R-Ras(wt) localization. Images were acquired at 2 min intervals for 20 mins and played at 7 frames/sec.

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Additional file 3:

Movie of GFP-R-Ras(38V) dynamics. Timelapse images of GFP-R-Ras(38V) localization. Images were acquired at 2 min intervals for 20 mins and played at 7 frames/sec.

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Additional file 4:

Movie of GFP-R-Ras(41A) dynamics. Timelapse images of GFP-R-Ras(41A) localization. Images were acquired at 2 min intervals for 20 mins and played at 7 frames/sec.

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Additional file 5:

GFP-R-Ras ruffle endocytosis. Montage of the GFP-R-Ras(wt) cell in Figure 2A. Two separate ruffles can be seen to disappear over time coincident with the appearance of vesicle-like structures at the site of the former ruffle. Images were acquired at 2 minute intervals for 30 minutes and shown at 3× magnification compared to the original.

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Additional file 6:

Actin dependence of intracellular vesicle traffic. Timelapse images of a GFP-R-Ras(41A) cell where the application of 30 mM 2,3-butanedione monoxime (BDM) to cells half way through the movie arrests dynamic intracellular movement. Images were acquired at 1 min intervals for 26 mins and played at 3 frames/sec.

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Additional file 7:

GFP-R-Ras is endocytosed to lysosomes via a non-caveolin-1 mediated pathway. (A-C) Shown are enlarged images of unfixed cells transfected with GFP-R-Ras constructs that were incubated with 1 μM Lysotracker Red DND-99 for 20 mins followed by washout. All Lysotracker-positive compartments larger than 1.2 μm contained R-Ras (n = 7 GFP-R-Ras(wt) cells, n = 8 GFP-R-Ras(38V) cells). There were numerous, smaller compartments in all cell types with no correlation between GFP-R-Ras and Lysotracker. (D-F) Transfected cells were fixed and stained for phospho-specific (Tyr-14) caveolin-1, but the lack of correspondence with GFP-R-Ras suggests that GFP-R-Ras is endocytosed separate from caveolin-1. Scale bar is 20 μm.

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Additional file 8:

Movie of R-Ras and β1-integrin vesicle trafficking-Inset. Timelapse images of GFP-Ras (green) and Alexa Fluor555 (red) labeled β1-integrin antibody. The movie is an inset of a GFP-R-Ras(wt)-expressing cell. Images were acquired at one minute intervals for 40 minutes and played at 7 frames/sec. Data was deconvolved using the no-neighbors algorithm.

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Additional file 9:

Movie of R-Ras and β1-integrin vesicle trafficking-Full view. Timelapse images of GFP-Ras (green) and Alexa Fluor555 (red) labeled β1-integrin antibody. The movie is of a GFP-R-Ras(wt)-expressing cell. Images were acquired at one minute intervals for 40 minutes and played at 7 frames/sec. Data was deconvolved using the no-neighbors algorithm.

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Additional file 10:

Movie of GFP-R-Ras(41A) and β1-integrin vesicle trafficking. Timelapse images of GFP-Ras (green) and Alexa Fluor555 (red) labeled β1-integrin antibody. The movie is of a GFP-R-Ras(41A)-expressing cell. Images were acquired at one minute intervals for 40 minutes and played at 7 frames/sec. Data was deconvolved using the no-neighbors algorithm.

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Additional file 11:

Movie of GFP-β1-integrin in membrane ruffles. Movie of GFP-β1-integrin transfected cells illustrate ruffling and endocytosis. Images were acquired 30 seconds apart for 30 mins. Data was deconvolved using the no-neighbors algorithm.

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Additional file 12:

Endocytosis of β1-integrin through the breakdown of ruffles. Inset view of movie 7 showing GFP-β1-integrin fluorescence breakdown into vesicles following ruffling. Images were acquired 30 seconds apart for 30 mins. Data was deconvolved using the no-neighbors algorithm.

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Additional file 13:

GFP-VSVG. Movie of a GFP-VSVG transfected cell with a ruffling membrane. Images were acquired 30 seconds apart for 30 mins. Data was deconvolved using the no-neighbors algorithm.

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Additional file 14:

R-Ras, β1-integrin, and VSVG colocalize within ruffles. Cells were transfected either with GFP-VSVG or GFP-β1-integrin, and then stained with anti-R-Ras or anti-β1-integrin antibody, as indicated. VSVG, R-Ras and β1-integrin all colocalize within ruffles (yellow). Scale bar = 20 μm.

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