Abstract
Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis.
Highlights
Endocytosis occurs by multiple means at the cell surface [1,2]
In addition to the clathrin-dependent (CD) endocytic pathway, numerous clathrin-independent endocytic mechanisms continue to be discovered [3]. These include a pinocytic pathway called the clathrin-independent endocytic carriers (CLICs)/GEEC (CG) pathway [4], which is responsible for internalizing a large fraction of the fluid phase, many GPIanchored proteins, and some specific cell surface proteins such as CD44 and G-protein coupled receptors [4,5,6]
Ultrastructural analyses show that this endocytic process is initiated by a pleomorphic set of clathrin-independent endocytic carriers (CLICs; [9]) that eventually fuse with distinct early endosomal compartments called GEECs (GPI-anchored protein enriched endosomes) [10,11]
Summary
In addition to the clathrin-dependent (CD) endocytic pathway, numerous clathrin-independent endocytic mechanisms continue to be discovered [3]. These include a pinocytic pathway called the CLIC/GEEC (CG) pathway [4], which is responsible for internalizing a large fraction of the fluid phase, many GPIanchored proteins, and some specific cell surface proteins such as CD44 and G-protein coupled receptors [4,5,6]. Membrane and volume components of these endosomal compartments are recycled to the cell surface [12,13], or addressed to a variety of cargo-specific locations [4] including the sorting endosome, which is the major recipient of cargo from the CD endocytic pathway [11]
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