Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.
We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.
Guha
A
A
National Center for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bangalore 560 065, India.
Sriram
V
V
Krishnan
K S
KS
Mayor
S
S
D000818:Animals; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D034281:Dynamins / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D006434:Hemocytes / Q000378:metabolism*; D007814:Larva / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D010873:Pinocytosis / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins
D029721:Drosophila Proteins; D017261:Glycosylphosphatidylinositols; D011956:Receptors, Cell Surface; C485128:anionic ligand-binding receptor, Drosophila; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins; C439628:shi protein, Drosophila