Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.

Abstract

Abstract. We have isolated and characterized temperature-sensitive 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 (FACS TM) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indyl) 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 indyl cells at the permissive temperature was poorly internalized in indyl 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 membranetrafficking 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 compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indyl cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indyl mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning.NDOCYTOSIS plays an important role in a wide variety of functions in eukaryotic cells, including nutrient uptake, internalization of receptors, antigen processing, and retrieval of membrane inserted into the cell surface during secretion. In animal cells, endocytosis typically involves the budding of clathrin-coated pits from the plasma membrane to yield coated vesicles that rapidly uncoat and fuse with early endosomes, a heterogeneous population of tubules and vesicles scattered throughout the cytoplasm. In early endosomes, internalized plasma membrane components and extracellular macromolecules are sorted such that proteins to be recycled back to the cell surface are separated from those destined for transport to late endosomes and lysosomes. A great deal is known about the kinetics and morphology of the intraceUular compartments involved in mammalian cells, but the molecular mechanisms for these various steps remain poorly understood.A number of proteins that play important roles during endocytosis have been identified. These include low molecular weight GTPases of the rab and ARF families, members of which have been implicated in regulating the formation or

Actin-, myosin- and ubiquitin-dependent endocytosis

“…It is possible that the same basic mechanism that has been proposed to function in phagocytosis and macropinocytosis also works in budding of uncoated micropinocytic vesicles. The signal transduction pathways that lead to activation of these two endocytic pathways are probably different, because D. discoideum mutants differentially affect them [73,74], but the molecular machinery involved seems to be very similar. Both phagocytosis and macropinocytosis are blocked in the presence of cytochalasin D [75,76], and the formation of the phagocytic cups as well as membrane ruffles and macropinocytic processes are associated with directed actin polymerization, and triggered by the activation of GTPases [76][77][78].…”

Circulation of the Plasma Membrane inDictyostelium

“…First, the earlier rates of membrane uptake determined in Ax2 suggested that one cell surface equivalent is taken up about every 45 min (Thilo and Vogel, 1980;Thilo, 1985;Bacon et al, 1994). Because Ax2 cells move one cell length in a few minutes, the earlier rates were incompatible with a membrane flow model for locomotion.…”

“…In an early report, the internalization of surface proteins labeled with radioactive galactose was followed; this suggested that an area equivalent to that of the plasma membrane is internalized once every 45 min (Thilo and Vogel, 1980;Thilo, 1985). A similar rate was subsequently found for a 120-kDa surface protein (Bacon et al, 1994). By contrast, the cyclic AMP receptor cAR1 appears to be confined to the cell surface and does not enter the cell (Xiao et al, 1997).…”

“…For a cylindrical Dictyostelium amoeba, this means an area equivalent to its surface for each cell length it moves, which it can do once every few minutes. In other words, the deduced rate of surface uptake of once each 45 min (Thilo and Vogel, 1980;Thilo, 1985;Bacon et al, 1994) implies that membrane flow models for how cells move could not apply to these cells. A similar conclusion comes from a comparison of NC4 and Ax3.…”

Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis inDictyostelium discoideum

“…Fluid-phase endocytosis and exocytosis of surface-attached wildtype Ax2, rescue cells, and dymA Ϫ cells were quantitatively analyzed using a method adapted from Bacon et al (1994). Cells were grown to confluency on 6-cm Petri dishes or in shaking culture at a density of 4 ϫ 10 6 cells/ml.…”