Coat proteins play multiple roles in the life cycle of a membrane-bound transport intermediate, functioning in lipid bilayer remodeling, cargo selection and targeting to an acceptor compartment. The Coat Protein complex II (COPII) coat is known to act in each of these capacities, but recent work highlights the necessity for numerous accessory factors at all stages of transport carrier existence. Here, we review recent findings that highlight the roles of COPII and its regulators in the biogenesis of tubular COPII-coated carriers in mammalian cells that enable cargo transport between the endoplasmic reticulum and ER-Golgi intermediate compartments, the first step in a series of trafficking events that ultimately allows for the distribution of biosynthetic secretory cargoes throughout the entire endomembrane system. K E Y W O R D S COPII coat, early secretory pathway, endoplasmic reticulum, ER exit site, posttranslational modification, Sec16A, Tango1/cTAGE5, TFG

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early-and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation. lattice light sheet microscopy | ESCRT microdomain | CRISPR/Cas9 | epidermal growth factor receptor | organelle

Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a transmembrane collagenase highly expressed in metastatic ovarian cancer and correlates with poor survival. Accumulating evidence shows that the cytoplasmic tail of MT1-MMP is subjected to phosphorylation, and this post-translational modification regulates enzymatic activity at the cell surface. To investigate the potential role of MT1-MMP cytoplasmic residue Thr phosphorylation in regulation of metastasis-associated behaviors, ovarian cancer cells that express low endogenous levels of MT1-MMP were engineered to express wild-type MT1-MMP, a phosphomimetic mutant (T567E), or a phosphodeficient mutant (T567A). Results show that Thr modulation influences behavior of both individual cells and multicellular aggregates (MCAs). The acquisition of either wild-type or mutant MT1-MMP expression results in altered cohesion of epithelial sheets and the formation of more compact MCAs relative to parental cells. Cells expressing MT1-MMP-T567E phosphomimetic mutants exhibit enhanced cell migration. Furthermore, MCAs formed from MT1-MMP-T567E-expressing cells adhere avidly to both intact peritoneal explants and three-dimensional collagen gels. Interaction of these MCAs with peritoneal mesothelium disrupts mesothelial integrity, exposing the submesothelial collagen matrix on which MT1-MMP-T567E MCAs rapidly disperse. Together, these findings suggest that post-translational regulation of the Thr in the MT1-MMP cytoplasmic tail may function as a regulatory mechanism to impact ovarian cancer metastatic success.

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