shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes
shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes
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DOI：
10.1242/jcs.00637
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发表时间：
2003-08-15
影响因子：
4
通讯作者：
Mayor, S
中科院分区：
文献类型：
--
作者：
Guha, A;Sriram, V;Mayor, S
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 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 shi(ts) hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.
