Computerized image-intensified fluorescence microscopy has been used to quantify routing and subcellular concentrations of rhodaminated EGF (Rh-EGF) during its receptor-mediated endocytosis in two transfected NIH-3T3 cell lines expressing 2 X 10(5) and 1.5 X 10(6) receptors per cell, respectively. A series of images were digitized by focusing at different depths through the volume of a single cell. The digitized pictures were corrected for fluorescence photobleaching, and removal of out-of-focus fluorescence contributions by deconvolution using the point spread function of the microscope optics (Agard, D. A., and J. W. Sedat. 1980. Proc. Soc. Photo-Opt. Instr. Eng. 264:110-117) allowed automatic computer analysis of the time dependence of endosomal vesicle size and fluorescence intensity in a live cell and also enabled the study of isolated vesicles. An increase in the amount of fluorescence bound to the cell surface, either by increasing the number of receptors expressed per cell or the concentration of Rh-EGF in the incubation drop, yielded an increase in the total fluorescence of internalized vesicles without an increase in their number and area. The linear relation between fluorescence intensity and area for vesicles at different times indicates that EGF concentration is conserved. This is compatible with fusion of small vesicles to form larger ones. However, as endocytosis proceeds, a twofold increase in the slope of the fluorescence vs. area plots is observed for larger vesicles, suggesting that active sorting causes the EGF to be concentrated. Alternatively, this factor could be produced by cumulative fluorescence contributions from stacked membranes. Since coated pits are internalized independent of their occupancy with EGF receptor, we propose that endocytosis does not involve a mechanism specifically recognizing occupied receptor but is rather triggered by a global intracellular event.
Skip Nav Destination
Article navigation
1 November 1989
Article|
November 01 1989
Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy.
M Benveniste,
M Benveniste
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Search for other works by this author on:
J Schlessinger,
J Schlessinger
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Search for other works by this author on:
Z Kam
Z Kam
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Search for other works by this author on:
M Benveniste
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
J Schlessinger
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Z Kam
Department of Polymer Research, Weizmann Institute of Science, Rehovot, Israel.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1989) 109 (5): 2105–2115.
Citation
M Benveniste, J Schlessinger, Z Kam; Characterization of internalization and endosome formation of epidermal growth factor in transfected NIH-3T3 cells by computerized image-intensified three-dimensional fluorescence microscopy.. J Cell Biol 1 November 1989; 109 (5): 2105–2115. doi: https://doi.org/10.1083/jcb.109.5.2105
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Sign in via your Institution
Sign in via your InstitutionSuggested Content
Email alerts
Advertisement
Advertisement