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Volume 5 issue 1January 2004
Stimulation-Dependent Recycling of Integrin 1 Regulated by ARF6 and Rab11
Aimee M. Powelka1,4,5, Jianlan Sun1,5, Jian Li1,5, Minggeng Gao1, Leslie M. Shaw2, Arnoud Sonnenberg3, and Victor W. Hsu1,6
1Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 USA
2Department of Pathology, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02115 USA
3Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
4Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115 USA
5Contributed equally to this work
6Address Correspondence to:
Victor Hsu
Brigham and Women's Hospital
One Jimmy Fund Way, Smith 538
Boston, MA 02115
Tel: 617-525-1103
FAX: 617-525-1104
Email: vhsu@rics.bwh.harvard.edu
Keywords: Endocytic recycling, ARF6, Rab11, integrin, cell motility
Running title: Integrin recycling regulated by ARF6 and Rab11
S1: Tracking the endocytic behaviour of Β1 through surface biotinylation. The biochemical recycling assay using biotin labeling was performed at different time points as indicated. Quantitation for the level of Β1 remaining internal after stimulation for recycling was performed at different time points with respect to the initial time point. Results are presented as a mean of two separate experiments with standard error.
S2: Surface Β1 in stimulated cells accumulates at the internal compartment similarly as in starved cells. HeLa cells were starved and the anti-integrin Β1 antibody TS1/26 was bound to the surface, followed by internalization for various times in the presence of different stimulants as indicated. Cells were then analyzed by immunofluorescence microscopy. Bar, 15 µm.
View S1 and 2
S3: The accumulation of surface Β1 at the recycling endosome is unaffected by dominant negative mutants of ARF6, Rab4, and Rab11. HeLa cells were transiently transfected with ARF6-T27N, Rab4-S22N, or Rab11-S25N. Antibody-bound Β1 at the cell surface were then allowed to internalize for 2 hours in starved cells. Cells were then fixed to examine Β1 (red) and small GTPases (green). Bar, 10 µm.
View S3
S4: Internalized Β1 in MDA-MB-231 cells undergoes stimulation-dependent recycling that is regulated by ARF6 and Rab11. (A) Stimulation-dependent recycling of internalized Β1 to peripheral ruffles. Image at 0 minutes shows the distribution of antibody-bound surface Β1 that has been internalized for 2 hours, while image at 5 minutes shows the distribution of this internalized Β1 after 5 minutes of stimulation. Bar, 15 µm. (B) Overexpression of ARF6-T27N inhibits the recycling of internal Β1 to surface membrane ruffles. MDA-MB-231 cells were transiently transfected with ARF6-T27N, and then antibody-bound surface was internalized for 2 hours under starvation condition. Cells were then stimulated for 5 minutes. Bar, 15 µm. (C) Overexpression of Rab11-S25N inhibits the recycling of internal Β1 to surface membrane ruffles. MDA-MB-231 cells were transiently transfected with Rab11-S25N, and then treated similarly as described in (B). Bar, 15 µm.
View S4
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