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.

Glucocorticoid mediated inhibition of LKB1 mutant non-small cell lung cancers.

The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.

Telehealth use by walk-in patients who subsequently visited an academic emergency department: a repeated cross-sectional study during the COVID-19 summer 2021.

AIM OF THE STUDY: During the COVID-19 pandemic, telehealth use increased worldwide in a variety of medical specialities and reached new population groups. A baseline survey of telehealth use prior to admission to the emergency department (ED) conducted before COVID-19 concluded that predominantly well-educated men used telehealth. It is unclear how COVID-19 changed the use of telehealth in Swiss emergency patients. We therefore aimed to investigate (i) the frequency of telehealth use during the pandemic, and (ii) how the pandemic has influenced telehealth use and users. MATERIALS AND METHODS: A repeated cross-sectional study was conducted among ED walk-in patients at a tertiary university hospital in Switzerland. The study took place one and a half years after the first confirmed COVID-19 case, during 30 shifts from 8 to 29 July 2021 and compared with the baseline survey conducted in 2019. Eligible patients were questioned about their use of, and attitudes to telehealth. RESULTS: A total of 1020 patients were screened for the COVID survey and 443 complete questionnaires were evaluated. A trend towards a general increase (+6.4%) in telehealth use was demonstrated (50.3%, n = 223 COVID survey vs 43.9%, n = 183 baseline survey; p = 0.058), with a shift to more female patients using telehealth in the COVID survey (female 54.9%, n = 124 vs 45.1%, n = 102; p = 0.052). During the pandemic, first use of telehealth was reported by 12.2% (n = 54) of patients, with a significant increase among patients with low educational status, and the latter patients often indicated that they did not plan to use telehealth after the pandemic. The perceived usefulness of telehealth and adherence to recommendations increased in the COVID survey compared with the baseline survey (adherence 90.3%, n = 149, vs 78.0%, n = 131; p = 0.002). CONCLUSION: We found a trend towards increased use of telehealth among Swiss ED patients. First-time users of telehealth were predominantly less educated and inclusion of these user groups may not be sustainable, as was indicated by the patients. COVID-19 led to greater adherence to telehealth recommendations and higher perceived usefulness. This could be due to the limited access to healthcare providers due to pandemic precautions. When offering telehealth, the needs of all patient groups must be considered, in order to ensure that telehealth provides the greatest benefit with lower barriers to use.

Did the pandemic influence telehealth use among Swiss emergency department patients? A sequential explanatory study.

OBJECTIVE: The aim of this study was to explore pandemic telehealth use among walk-in emergency department (ED) patients at Bern University Hospital. DESIGN: As in sequential explanatory designs, quantitative data were collected first. To explain the quantitative results, telehealth use was explored qualitatively using an interview guide informed by the quantitative results. SETTING: The University Hospital of Bern ED designed a follow-up cross-sectional study (baseline done in 2019) to assess telehealth use among ED walk-in patients during the pandemic (2021). PARTICIPANTS: We included participants of all age groups that had consented to a follow-up qualitative study and also ensured a gender and age balance. We aimed for data saturation that was achieved by the seventh key informant. A total of 11 key informants took part in the study. RESULTS: Three main themes emerged, namely: (1) telehealth use means the use of a telephone for many; (2) telehealth has both remits and limits; and (3) perceived future telehealth opportunities and threats. CONCLUSION: The pandemic seems not to have increased telehealth use among walk-in ED patients. The slight increase observed in telehealth use among women seems related to the use of the COVID-19 app from trusted sites like the Federal Office of Public Health. Telehealth emerged as having remits, limits, opportunities and threats. The human factor preference emerged as very important to all key informants. The fear that telehealth threatens the human factor cannot be over emphasised. The telephone remains the biggest telehealth modality among Swiss ED walk-in patients.

Targeted delivery of a vaccine protein to Langerhans cells in the human skin via the C-type lectin receptor Langerin.

Human skin is a preferred vaccination site as it harbors multiple dendritic cell (DC) subsets, which display distinct C-type lectin receptors (CLR) that recognize pathogens. Antigens can be delivered to CLR by antibodies or ligands to boost antigen-specific immune responses. This concept has been established in mouse models but detailed insights into the functional consequences of antigen delivery to human skin DC in situ are sparse. In this study, we cloned and produced an anti-human Langerin antibody conjugated to the EBV nuclear antigen 1 (EBNA1). We confirmed specific binding of anti-Langerin-EBNA1 to Langerhans cells (LC). This novel LC-based vaccine was then compared to an existing anti-DEC-205-EBNA1 fusion protein by loading LC in epidermal cell suspensions before coculturing them with autologous T cells. After restimulation with EBNA1-peptides, we detected elevated levels of IFN-γ- and TNF-α-positive CD4+ T cells with both vaccines. When we injected the fusion proteins intradermally into human skin explants, emigrated skin DC targeted via DEC-205-induced cytokine production by T cells, whereas the Langerin-based vaccine failed to do so. In summary, we demonstrate that antibody-targeting approaches via the skin are promising vaccination strategies, however, further optimizations of vaccines are required to induce potent immune responses.

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.

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.

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.

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.

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.

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.

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.