CCDC32 stabilizes clathrin-coated pits and drives their invagination.

Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. Quantitative live cell imaging reveals that CCDC32, a poorly characterized endocytic accessory protein, regulates CCP stabilization and is required for efficient CCP invagination. CCDC32 interacts with the α-appendage domain (AD) of AP2 via its coiled-coil domain to exert this function. Furthermore, we showed that the clinically observed nonsense mutations in CCDC32, which result in the development of cardio-facio-neuro-developmental syndrome (CFNDS), inhibit CME by abolishing CCDC32-AP2 interactions. Overall, our data demonstrates the function and molecular mechanism of a novel endocytic accessory protein, CCDC32, in CME regulation. Significance Statement: Clathrin-mediated endocytosis (CME) happens via the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). In this study, we used a combination of quantitative live cell imaging, ultrastructure electron microscopy and biochemical experiments to show that CCDC32, a poorly studied and functional ambiguous protein, acts as an important endocytic accessory protein that regulates CCP stabilization and invagination. Specifically, CCDC32 exerts this function via its interactions with AP2, and the coiled-coil domain of CCDC32 and the α-appendage domain (AD) of AP2 are essential in mediating CCDC32-AP2 interactions. Importantly, we demonstrate that clinically observed loss-of-function mutations in CCDC32 lose AP2 interaction capacity and inhibit CME, resulting in the development of cardio-facio-neuro-developmental syndrome (CFNDS).

Endocytosis gated by emergent properties of membrane-clathrin interactions.

Clathrin-mediated endocytosis (CME), the major cellular entry pathway, starts when clathrin assembles on the plasma membrane into clathrin-coated pits (CCPs). Two populations of CCPs are detected within the same cell: 'productive' CCPs that invaginate and pinch off, forming clathrin-coated vesicles (CCVs) [1, 2], and 'abortive' CCPs [3, 4, 5] that prematurely disassemble. The mechanisms of gating between these two populations and their relations to the functions of dozens of early-acting endocytic accessory proteins (EAPs) [5, 6, 7, 8, 9] have remained elusive. Here, we use experimentally-guided modeling to integrate the clathrin machinery and membrane mechanics in a single dynamical system. We show that the split between the two populations is an emergent property of this system, in which a switch between an Open state and a Closed state follows from the competition between the chemical energy of the clathrin basket and the mechanical energy of membrane bending. In silico experiments revealed an abrupt transition between the two states that acutely depends on the strength of the clathrin basket. This critical strength is lowered by membrane-bending EAPs [10, 11, 12]. Thus, CME is poised to be shifted between abortive and productive events by small changes in membrane curvature and/or coat stability. This model clarifies the workings of a putative endocytic checkpoint whose existence was previously proposed based on statistical analyses of the lifetime distributions of CCPs [4, 13]. Overall, a mechanistic framework is established to elucidate the diverse and redundant functions of EAPs in regulating CME progression.

Sorting nexin 5 mediates virus-induced autophagy and immunity.

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Evolving models for assembling and shaping clathrin-coated pits.

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics.

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby-CLCa and/or µ2-eGFP, σ2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α-eGFP). Using biochemical and TIRFM-based assays, we compared the functionality of the AP2 markers. All of the eGFP-tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image-based CCP analyses than mRuby-CLCa. However, overexpression of either µ2-eGFP or σ2-eGFP impaired transferrin receptor uptake. In addition, µ2-eGFP reduced the rates of CCP initiation and σ2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α-eGFP. Moreover, α-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes α-eGFP as a robust, fully functional marker for CME.

A functionally neutral single chain antibody to measure beta-1 integrin uptake and recycling.

Integrin-mediated cell adhesion and signaling are critical for many physiological processes. The dynamic turnover of integrins and their associated adhesion complexes through endocytic and recycling pathways has emerged as an important mechanism for controlling cell migration and invasion in cancer. Thus, the regulation of integrin trafficking and how this may be altered by disease-specific molecular mechanisms has generated considerable interest. However, current tools available to study integrin trafficking may cause artifacts and/or do not provide adequate kinetic information. Here, we report the generation of a functionally neutral and monovalent single chain antibody to quantitatively and qualitatively measure ß1 integrin trafficking in cells. Our novel probe can be used in a variety of assays and allows for the biochemical characterization of rapid recycling of endogenous integrins. We also demonstrate its potential utility in live cell imaging, providing proof of principle to guide future integrin probe design.

FCHSD2 controls oncogenic ERK1/2 signaling outcome by regulating endocytic trafficking.

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.

Wbox2: A clathrin terminal domain-derived peptide inhibitor of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane-permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.

Early and nonredundant functions of dynamin isoforms in clathrin-mediated endocytosis.

Dynamin GTPases (Dyn1 and Dyn2) are indispensable proteins of the core clathrin-mediated endocytosis (CME) machinery. Best known for their role in fission at the late stages of CME, many studies have suggested that dynamin also plays a regulatory role during the early stages of CME; however, detailed studies regarding isoform-specific early regulatory functions of the dynamins are lacking. With a recent understanding of the regulation of Dyn1 in nonneuronal cells and improved algorithms for highly sensitive and quantitative analysis of clathrin-coated pit (CCP) dynamics, we have evaluated the differential functions of dynamin isoforms in CME using domain swap chimeras. We report that Dyn1 and Dyn2 play nonredundant, early regulatory roles during CME in nonneuronal cells. The proline/arginine-rich domain of Dyn2 is important for its targeting to nascent and growing CCPs, whereas the membrane-binding and curvature-generating pleckstrin homology domain of Dyn1 plays an important role in stabilizing nascent CCPs. We confirm the enhanced ability of dephosphorylated Dyn1 to support CME, even at substoichiometric levels compared with Dyn2. Domain swap chimeras also revealed previously unknown functional differences in the GTPase and stalk domains. Our study significantly extends the current understanding of the regulatory roles played by dynamin isoforms during early stages of CME.

DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Mutant p53 amplifies a dynamin-1/APPL1 endosome feedback loop that regulates recycling and migration.

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors downstream of gain-of-function (GOF) mutant p53. We report that dynamin-1 (Dyn1) is up-regulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Dyn1 is required for the recruitment and accumulation of the signaling scaffold, APPL1, to a spatially localized subpopulation of endosomes at the cell perimeter. We developed new tools to quantify peripherally localized early endosomes and measure the rapid recycling of integrins. We report that these perimeter APPL1 endosomes modulate Akt signaling and activate Dyn1 to create a positive feedback loop required for rapid recycling of EGFR and ß1 integrins, increased focal adhesion turnover, and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus that integrates signaling and receptor trafficking, which can be co-opted and amplified in mutant p53-driven cancer cells to increase migration and invasion.

A nostalgic look back 40 years after the discovery of receptor-mediated endocytosis.

The concept of receptor-mediated endocytosis was proposed 40 years ago in a seminal review by Joseph Goldstein, Michael Brown, and Richard Anderson. Not only their hypothesis but also the lessons learned that guided their discovery have stood the test of time. I recount some of these herein, while also looking back nostalgically at a forgotten era of scientific communication.

Role for ERK1/2-dependent activation of FCHSD2 in cancer cell-selective regulation of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) regulates the uptake of cell-surface receptors as well as their downstream signaling activities. We recently reported that signaling can reciprocally regulate CME in cancer cells and that this crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME across a panel of normal and cancerous cells. Inhibition of several kinases selectively affected CME in cancer cells, including inhibition of ERK1/2, which selectively inhibited CME by decreasing the rate of clathrin-coated pit (CCP) initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. Our data suggest that ERK1/2 phosphorylation activates FCHSD2 and regulates EGF receptor (EGFR) endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in nonsmall cell lung cancer (NSCLC) cells leads to increased cell-surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher NSCLC patient survival rates, suggesting that FCHSD2 can negatively affect cancer progression. These findings provide insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.

The dynamin superfamily.

The dynamin superfamily comprises a growing assortment of multi-domain GTPases, found from bacteria to man, that are distinguished from typical GTPases of the Ras, Rab and G-protein families by their modular structure (Figure 1), relatively large size (>70 kDa), and low affinity for guanine nucleotides. In addition, they display a conserved propensity to self-assemble into polymeric arrays, the dynamics of which are regulated by an autonomous, assembly-stimulated GTPase activity.

A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells.

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 (GSK3ß) 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 GSK3ß 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.

Regulation of Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.