Focal adhesion motility revealed in stationary fibroblasts

Science

Volumn 286, Issue 5442, 1999, Pages 1172-1174

  Smilenov, Lubomir B ^a   Mikhailov, Alexei ^a,b   Pelham Jr , Robert J ^a   Marcantonio, Eugene E ^a   Gundersen, Gregg G ^a  

^a Och Spine at New York Presbyterian Hospitals   (United States)

^b MASSACHUSETTS GENERAL HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ACTIN; FOCAL ADHESION KINASE; GREEN FLUORESCENT PROTEIN; INTEGRIN;

ACTIN FILAMENT; ANIMAL CELL; ARTICLE; CELL ADHESION; CELL MIGRATION; CELL STRAIN 3T3; MOUSE; NONHUMAN; PRIORITY JOURNAL;

3T3 CELLS; ACTINS; ANIMALS; ANTIGENS, CD29; CELL ADHESION; CELL COUNT; CELL LINE; CELL MOVEMENT; FIBROBLASTS; FLUORESCENCE; GREEN FLUORESCENT PROTEINS; LUMINESCENT PROTEINS; MICE; MICROSCOPY, INTERFERENCE; RATS; RECOMBINANT FUSION PROTEINS;

ANIMALIA;

EID: 0033527692     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5442.1172     Document Type: Article

References (24)

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9. NIH 3T3 cells were stably transfected with pLen GFP-β1 (14) and pSVneo as described (5). Cell lines expressing low levels of GFP-β1 integrin were characterized by flow cytometry using methods described in (5). We confirmed that GFP was extracellular by labeling with antibodies to GFP.
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19. Cells were assembled into chambers and imaged as described (15) using a Nikon Eclipse TE300 microscope maintained at 37°C. A Nikon HQ fluorescein filter cube was used for GFP fluorescence. Fluorescence and phase-contrast images were recorded with a cooled charge-coupled device (CCD) camera with a back-illuminated chip (Princeton Instruments, Trenton, NJ) and Metamorph software. Sequential images were taken every 1 to 4 min.
20. Cells were plated on fibronectin-coated cover slips, fixed, and stained as described (5). Rabbit antibody to GFP was from Clontech, mouse antibody to integrin α5 was from Pharmingen, and anti-vinculin was purchased from Sigma. The secondary antibodies were from Cappel (Durham, NC).
21. For movies, see Science Online (www.sciencemag. org/feature/data.1042403.shl).
22. R-actin (2 mg/ml; Cytoskeleton, Denver, CO) was microinjected into cells expressing GFP-integrin and allowed to incorporate for 2 to 4 hours before recording simultaneous GFP and rhodamine fluorescence (16). Sequential images were taken every 4 to 8 min.
23. IRM was done using a Nikon 100× Fluor objective with an iris. A green filter was used to eliminate higher-ordered reflections and to prevent excitation of GFP. IRM and GFP fluorescence images were recorded consecutively with the cooled CCD once every minute as described (16).
24. We thank T. Salmon and S. Inoue for stimulating discussions, J. Larson and P. Bent (Nikon) for help with microscopes, and R. Y. Tsien for S65T GFP. A.M. and R.J.P. were supported by National Institute on Aging and National Institute of General Medical Sciences training grants, respectively. Some experiments were done while G.G.G. was a Nikon Fellow at the Marine Biological Laboratories in Woods Hole, MA. Supported by NIH grants GM44585 (E.E.M.) and GM42026 (G.G.G.).