CSM News
Electronic Edition
Volume 3, number 5
July 29, 1994

Please submit abstracts of your papers as soon as they have been
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===========
 Abstracts
===========

Dictyostelium discoideum mutants with temperature-sensitive defects in
endocytosis

Rebecca A. Bacon, Christopher J. Cohen, David A. Lewin, and Ira Mellman

Department of Cell Biology, Yale University School of Medicine, New Haven, 
Connecticut 06520-8002

Journal of Cell Biology, in press

Abstract

   We have isolated and characterized temperature sensitive (ts)
endocytosis mutants in Dictyostelium discoideum.  Dictyostelium is an
attractive model for genetic studies of endocytosis because of its
high rates of endocytosis, its reliance on endocytosis for nutrient
uptake, and tractable molecular genetics.  Endocytosis-defective
mutants were isolated by a fluorescence-activated cell sorting as
cells unable to take up a fluorescent marker.  One ts mutant (indy1)
was characterized in detail and found to exhibit a complete block in
fluid phase endocytosis at the restrictive temperature but normal
rates of endocytosis at the permissive temperature.  Likewise, a
potential cell surface receptor that was rapidly internalized in wild
type cells and indy1 cells at the permissive temperature was poorly
internalized in indy1 under restrictive conditions.
 Growth was also completely arrested at the restrictive temperature.  The 
endocytosis block was rapidly induced upon shift to the restrictive 
temperature and reversed upon return to normal conditions.  Inhibition of 
endocytosis was also specific, as other membrane trafficking events such as 
phagocytosis, secretion of lysosomal enzymes, and contractile vacuole 
function were unaffected at the restrictive temperature.  Because recycling 
and transport to late endocytic coompartments were not affected, the site 
of action of the defect is probably at an early step in the endocytic 
pathway.  Additionally, indy1 cells were unable to proceed through the 
normal development program at the restrictive temperature.  Given the tight 
functional and growth phenotypes, the indy1 mutant provides an opportunity 
to isolate genes responsible for endocytosis in Dictyostelium by 
complementation cloning.

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