Diffusional mobility of Golgi proteins in membranes of living cells

Science

Volumn 273, Issue 5276, 1996, Pages 797-801

  Cole, Nelson B ^a   Smith, Carolyn L ^b   Sciaky, Noah ^a,e   Terasaki, Mark ^c   Edidin, Michael ^d   Lippincott Schwartz, Jennifer ^a  

^a EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT   (United States)

^b NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE   (United States)

^c UNIVERSITY OF CONNECTICUT HEALTH CENTER   (United States)

^d JOHNS HOPKINS UNIVERSITY   (United States)

^e NATIONAL JEWISH MEDICAL AND RESEARCH CENTER   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHIMERIC PROTEIN; MEMBRANE PROTEIN;

ANIMAL CELL; ARTICLE; BLEACHING; CONTROLLED STUDY; DIFFUSION; FLUORESCENCE; GOLGI COMPLEX; HELA CELL; HUMAN; HUMAN CELL; MAMMAL; MEMBRANE TRANSPORT; NONHUMAN; PRIORITY JOURNAL; PROTEIN IMMOBILIZATION; PROTEIN TARGETING; PROTEIN TRANSPORT;

AEQUOREA VICTORIA; ANIMALIA; MAMMALIA; VICTORIA;

EID: 0029784190     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5276.797     Document Type: Article

References (42)

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21. Energy depletion was performed as described [J. Donaldson et al., J. Cell. Biol. 111, 2295 (1990)]. This treatment dissociates coatomer complexes (which are believed to mediate vesicle traffic) from Golgi membranes of living cells, blocks export of proteins out of the ER, and prevents processing of newly synthesized proteins by Golgi enzymes. For further discussion of the inhibitory effects of energy depletion and reduced temperature on vesicle traffic, see C. J. Beckers et al., Cell 50, 523 (1987) and E. Kuismanen and J. Saraste, Methods Cell Biol. 32, 257 (1989).
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40. The microscope system described in (15) was used in the quantitative FPR experiments. The FPR beam was imaged into the sample as a stripe 2 μm wide. Because the stripe extended across the entire width of the Golgi or ER and bleached through the whole depth, diffusion was into and out of a line bounded on its sides, and not on its end. Hence, recovery of fluorescence was due to one-dimensional diffusion. The imposition of one-dimensional geometry on a complicated membrane as well as the mathematics for this case are covered in C-L. Wey, M. A. Edidin, R. A. Cone, Biophys. J. 33, 225 (1981). Briefly, a tortuous diffusion path reduces the apparent D, so our measurements in that case would be an underestimation.
41. 4 precipitation. Fluorescent cells were imaged at 37°C in buffered medium with a Zeiss LSM 410 confocal microscope system having a 100× Zeiss planapo objective (NA 1.4). The GFP molecule was excited with the 488 line of a krypton-argon laser and imaged with a 515-540 bandpass filter. Images were transferred to a Macintosh computer for editing and were printed with a Fujix Pictrography 3000 Digital Printer.
42. We thank R. Klausner, E. Siggia, J. Bonifacino, J. Zimmerberg, J. Donaldson, J. Presley, J. Ellenberg, and K. Zaal for valuable comments and suggestions, and M. Chalfie, K. Moremen, M. Fukuda, R. Poljak, and V. Hsu for generous gifts of reagents. M.E. is supported by grant R37 AI 14584. Quicktime movies are available at http://www.uchc.edu/htterasaki/ flip.html.