Regulation of endocytic clathrin dynamics by cargo ubiquitination.
ABSTRACT: VIDEO ABSTRACT:Some endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that ?-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor's first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached.
Project description:Clathrin-mediated endocytosis has long been viewed as a process driven by core endocytic proteins, with internalized cargo proteins being passive. In contrast, an emerging view suggests that signaling receptor cargo may actively control its fate by regulating the dynamics of clathrin-coated pits (CCPs) that mediate their internalization. Despite its physiological implications, very little is known about such "cargo-mediated regulation" of CCPs by signaling receptors. Here, using multicolor total internal reflection fluorescence microscopy imaging and quantitative analysis in live cells, we show that the ?-opioid receptor, a physiologically relevant G protein-coupled signaling receptor, delays the dynamics of CCPs in which it is localized. This delay is mediated by the interactions of two critical leucines on the receptor cytoplasmic tail. Unlike the previously known mechanism of cargo-mediated regulation, these residues regulate the lifetimes of dynamin, a key component of CCP scission. These results identify a novel means for selectively controlling the endocytosis of distinct cargo that share common trafficking components and indicate that CCP regulation by signaling receptors can operate via divergent modes.
Project description:Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2. FCHo2 assembles at the rim of CCPs to control CCP growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly. Our data suggest a mechanism for the nanoscale control of CCP growth and stability that may similarly apply to other metastable structures in cells.
Project description:Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. We have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. Our results show that influenza can exploit clathrin-mediated and clathrin- and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral-binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.
Project description:Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with Dyn2 but inactivated in non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3?) kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual- and triple-channel total internal reflection fluorescence microscopy, we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2 but not Dyn1 exhibits a pronounced burst of assembly, presumably into supramolecular collar-like structures that drive membrane scission and clathrin-coated vesicle (CCV) formation. Activation of Dyn1 by acute inhibition of GSK3? results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation, and decreased CCP lifetimes but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME that occur well upstream of fission, even when present at low, substoichiometric levels relative to Dyn2. Under physiological conditions, Dyn1 is activated downstream of epidermal growth factor receptor (EGFR) signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoform-specific regulatory role for Dyn1 in CME.
Project description:Clathrin-mediated endocytosis (CME) is the most characterized pathway for the endocytic entry of proteins and lipids at the plasma membrane of eukaryotic cells. Numerous studies have probed the roles of different endocytic accessory proteins in regulating the dynamics of clathrin-coated pit (CCP) assembly. However, it is not completely clear how physical cues regulate CCP dynamics. Here we employ microcontact printing to control cell shape and examine CCP dynamics as a function of cell spreading area for three differently sized cells. Cells with a large spreading area had more short-lived CCPs but a higher CCP initiation rate. Interestingly, we found that fluorescence intensity of CCPs decreased with increasing cell spreading area in a manner that was dependent on the cortical actin network. Our results point to another facet of the regulation of CCP dynamics, suggesting that CME may be modulated while cells change their mechanical state and remodel their actin cytoskeleton during various processes.
Project description:Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.
Project description:A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi-channel image acquisition and automated analysis of CCP dynamics in combination with a new method to quantify the time courses of recruitment of endocytic factors to CCPs of different lifetimes. Using this approach we have extracted the kinetics of recruitment and disassembly of fluorescently labeled clathrin and/or AP-2 throughout the entire lifetime of temporally defined CCP cohorts. On the basis of these analyses, we can (i) directly correlate recruitment profiles of these two proteins; (ii) define five distinct CCP maturation phases, i.e. initiation, growth, maturation, separation and departure; (iii) distinguish events with absolute versus fractional timing and (iv) provide information on the spatial distribution of fluorophores during CCP maturation. Emerging from these analyses is a more clearly defined role for AP-2 in determining the temporal hierarchy for clathrin recruitment and CCP maturation. This method provides a new means to identify other such hierarchies during CCP maturation.
Project description:Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.
Project description:Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP(2) synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5'-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP(2) synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.
Project description:Phosphoinositides are thought to play an important role in clathrin-coated pit (CCP) dynamics. Biochemical and structural studies have shown a direct interaction of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] with endocytic clathrin adaptors, whereas functional studies using cell-free systems or intact cells have demonstrated the importance of PI(4,5)P2 synthesis and dephosphorylation in clathrin coating and uncoating, respectively. Furthermore, genetic manipulations of kinases and phosphatases involved in PI(4,5)P2 metabolism result in major defects in synaptic vesicle recycling and other forms of clathrin-dependent endocytosis. However, live imaging studies of these enzymes at CCPs have not been conducted. We have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatial-temporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner endophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs. The 145-kDa isoform, the predominant isoform expressed in the brain, was rapidly recruited as a "burst," together with endophilin, at a late stage of CCP formation. In contrast, the nonneuronal ubiquitously expressed 170-kDa isoform of SJ1 was present at all stages of CCP formation. These results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP.