Clathrin-mediated endocytosis is the major mechanism for eukaryotic plasma membrane-based proteome turn-over. In plants, clathrin-mediated endocytosis is essential for physiology and development, but the identification and organization of the machinery operating this process remains largely obscure. Here, we identified an eight-core-component protein complex, the TPLATE complex, essential for plant growth via its role as major adaptor module for clathrin-mediated endocytosis. This complex consists of evolutionarily unique proteins that associate closely with core endocytic elements. The TPLATE complex is recruited as dynamic foci at the plasma membrane preceding recruitment of adaptor protein complex 2, clathrin, and dynamin-related proteins. Reduced function of different complex components severely impaired internalization of assorted endocytic cargoes, demonstrating its pivotal role in clathrin-mediated endocytosis. Taken together, the TPLATE complex is an early endocytic module representing a unique evolutionary plant adaptation of the canonical eukaryotic pathway for clathrin-mediated endocytosis.

SUMMARY High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach—profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes—pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Export of proteins from the endoplasmic reticulum (ER) in COPII-coated vesicles occurs at defined sites, which contain the scaffolding protein Sec16. We identify TFG-1, a new conserved regulator of protein secretion that interacts directly with SEC-16 and controls the export of cargoes from the ER in C. elegans. Hydrodynamic studies indicate that TFG-1 forms hexamers, which facilitate the co-assembly of Sec16 with COPII subunits. Consistent with these findings, TFG-1 depletion leads to a dramatic decline in both SEC-16 and COPII levels at ER exit sites. The amino-terminus of human TFG was identified previously as a fusion partner of two protein kinases, creating a pair of oncogenes. We propose that fusion of these kinases to TFG relocalizes their activities to ER exit sites, where they prematurely phosphorylate substrates during ER export. Our findings provide a mechanism by which translocations involving TFG can result in cellular transformation and oncogenesis.

Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway.clathrin | multivesicular endosome

The septins, but not midbody microtubules, are important for daughter cell cytoplasmic isolation and ESCRT-dependent midbody ring release during abscission.