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First published online July 10, 2003
doi: 10.1242/10.1242/jcs.00637

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shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes

A. Guha^1,2,*, V. Sriram^1,*, K. S. Krishnan^2, and S. Mayor^1,

¹ National Center for Biological Sciences, Tata Institute of Fundamental Research, GKVK, Bangalore 560 065, India
² Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhaba Road, Bombay 400 005, India

Authors for correspondence (e-mail: ksk{at}tifr.res.in; mayor{at}ncbs.res.in)

Accepted 28 April 2003

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 (shi^ts) parallels the temperature-sensitive behavior of shi^ts animals. At the restrictive temperature in shi^ts cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and -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 shi^ts hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.

Key words: Fluid phase, GPI-anchored proteins, Scavenger receptor, Endocytosis, Cell culture, shibire

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