Biology subjects

Schmid, S. L.

Publications and source records attributed to Schmid, S. L..

12 recordsLinked to original sources

bioRxiv2025

VAMP8 function reveals tight linkage between endocytic recycling and endocytosis

Clathrin-mediated endocytosis (CME) is a multistage process that involves the initiation and stabilization of clathrin-coated pits (CCPs) that invaginate and finally detach from the plasma membrane to form clathrin-coated vesicles (CCVs). Given that SNARE proteins are essential for downstream vesicle targeting and fusion events, their recruitment into nascent CCVs has been suggested to be a prerequisite for CME progression. However, which and how SNARE proteins regulate CME remains to be explored. Here, we showed that siRNA-mediated knockdown of the R-SNARE, VAMP8 impairs CCP initiation, stabilization and invagination and strongly inhibits CME. Mechanistically, recruitment of VAMP8 to CCVs is not required for CME. Instead, depletion of VAMP8 inhibits recycling of endocytic cargoes and as exemplified here by transferrin receptor, skews their trafficking toward lysosomal degradation. VAMP8 depletion therefore indirectly impairs CCV formation and inhibits CME by depleting endocytic cargo. Overall, our study provides new insights into the crosstalk between endocytosis and endocytic recycling of CME cargo and demonstrates the critical role for cargo recruitment in stabilizing nascent CCPs to regulate CME. Significance StatementWe demonstrate that VAMP8 depletion inhibits recycling of endocytic cargo and reroutes transferrin receptors toward lysosomal degradation, thereby impairing endocytic vesicle formation and inhibiting clathrin-mediated endocytosis. Thus, the work uncovers a critical link between endocytic recycling and endocytosis, highlighting the importance of recycling pathways in maintaining membrane dynamics. However, the recruitment of VAMP8 into nascent clathrin-coated pits by CALM is not required for CME. While SNARE recruitment is essential for formation of COPI and COPII vesicles, our findings suggest that this requirement is not generalizable to endocytic CCVs.

cell biology↗

bioRxiv2025

Dynamic early recruitment of GAK-Hsc70 regulates coated pit maturation

Clathrin-mediated endocytosis (CME) is the process by which clathrin assembles on the plasma membrane to form clathrin-coated pits (CCPs), which then invaginate, accumulate cargo and are released by fission from the membrane to form clathrin-coated vesicles (CCVs). A transition of nascent CCPs from flat-to-curved has been observed by various methods. However, what drives this transition remains unknown and controversial. GAK and its chaperone protein, Hsc70, are well-known to mediate clathrin release from CCVs and several studies have observed a late burst of GAK recruitment as CCVs form. Other studies have proposed that early recruitment of GAK-Hsc70 could function to provide the necessary energy source to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature and invagination of the growing CCP; however, direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. Furthermore, mutations in the J domain of GAK that abolish Hsc70 recruitment to and activation at CCPs, lead to the accumulation of GAK at CCPs, hinder CCP stabilization and invagination and result in a striking increase in the proportion of short-lived, abortive CCPs. These findings support the hypothesis that GAK-Hsc70 promotes the turnover and remodeling of nascent clathrin assemblies required for curvature development during CME. Significance StatementGAK and its chaperone protein, Hsc70, are known to be recruited to clathrin-coated vesicles (CCVs) to mediate clathrin uncoating. Previous studies have proposed that early recruitment of GAK-Hsc70 to CCPs could function to remodel nascent flat clathrin lattices, replacing hexagons with pentagons and enabling a gain of curvature of the assembling polymeric coat. However, there are conflicting views, and direct functional evidence is lacking. Here we show that GAK knockdown inhibits CCP formation and invagination. A detailed domain-specific mutational analysis of GAK pinpointed the importance of J domain-Hsc70 interactions in regulating crucial early steps of CME including CCP stabilization and invagination. These findings support a hypothesis that GAK-Hsc70 promotes turnover of clathrin at nascent CCPs required for curvature development.

cell biology↗

bioRxiv2024

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. We show by quantitative live cell imaging that siRNA-mediated knockdown of CCDC32, a poorly characterized endocytic accessory protein, leads to the accumulation of unstable flat clathrin assemblies. CCDC32 interacts with the -appendage domain (AD) of AP2 in vitro and with full length AP2 complexes in cells. Deletion of aa78-98 in CCDC32, corresponding to a predicted -helix, abrogates AP2 binding and CCDC32s early function in CME. Furthermore, clinically observed nonsense mutations in CCDC32, which result in C-terminal truncations that lack aa78-98, are linked to the development of cardio-facio-neuro-developmental syndrome (CFNDS). Overall, our data demonstrate the function of a novel endocytic accessory protein, CCDC32, in regulating CCP stabilization and invagination, critical early stages of CME. SummaryWe show that CCDC32, a poorly studied and functionally ambiguous protein, binds to AP2 and regulates CCP stabilization and invagination. Clinically observed mutations in CCDC32 lose their ability to interact with AP2 likely contributing to the development of cardio-facio-neuro-developmental syndrome.

cell biology↗

bioRxiv2023

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.

biophysics↗

bioRxiv2020

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-clathrin 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 unambiguously separates ACs from CCPs without an additional marker. 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 orthogonal to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering the function of individual EAPs.

cell biology↗

bioRxiv2020

FCHSD2 Controls Oncogenic ERK1/2 Signaling Outcome by Regulating Endocytic Trafficking

Cancer progression is driven, in part, by altered signaling downstream of receptor tyrosine kinases (RTKs). Surface expression and RTK activity are regulated by clathrin-mediated endocytosis (CME), endosomal recycling or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking, creating feedback loops that enhance tumor progression. We previously reported a cancer-cell specific function of FCHSD2 (FCH/F-BAR and double SH3 domain-containing protein) in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of EGFR and MET, diverting their trafficking toward late endosomes and lysosomes. FCHSD2 depletion results in the nuclear translocation of active ERK1/2, leading to enhanced transcription and upregulation of EGFR and MET. The small GTPase, Rab7, is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates with 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.

cell biology↗

bioRxiv2020

Structure-based inhibitors reveal roles for the clathrin terminal domain and its W-box binding site in CME

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 (TD), 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 TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis. SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

cell biology↗

bioRxiv2019

The mechanisms of dynamin-actin interaction

Cell-cell fusion is an indispensable process in the conception, development and physiology of multicellular organisms. Here we demonstrate a direct and noncanonical role for dynamin, best known as a fission GTPase in endocytosis, in cell-cell fusion. Our genetic and cell biological analyses show that dynamin colocalizes within the F-actin-enriched podosome-like structures at the fusogenic synapse, which is required for generating invasive membrane protrusions and myoblast fusion in vivo, in an endocytosis-independent manner. Biochemical, negative stain EM and cryo-electron tomography (cryo-ET) analyses revealed that dynamin forms helices that directly bundles actin filaments by capturing multiple actin filaments at their outer rim via interactions with dynamins proline-rich domain. GTP hydrolysis by dynamin triggers disassembly of the dynamin helix, exposes the sides of the actin filaments, promotes dynamic Arp2/3-mediated branched actin polymerization, and generates a mechanically stiff actin network. Thus, dynamin functions as a unique actin-bundling protein that enhances mechanical force generation by the F-actin network in a GTPase-dependent manner. Our findings have universal implications for understanding dynamin-actin interactions in various cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2018

Mutant p53 triggers a dynamin-1/APPL1 endosome feedback loop that regulates β1 integrin recycling and migration

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors. We report that gain-of-function (GOF) mutant p53 expression enhances {beta} integrin and EGF receptor recycling and increases cell migration by triggering a positive feedback loop involving the activation of dynamin-1 (Dyn1) and accumulation of a spatially-restricted subpopulation of APPL1-positive perimeter endosomes. DNM1 is upregulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Perimeter APPL1 endosomes are required for rapid recycling of EGFR and {beta}1 integrins and modulate Akt signaling and Dyn1 activation to create the positive feedback loop that culminates in increased focal adhesion turnover and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus, integrating signaling and receptor trafficking, that can be co-opted by cancer cells for mutant p53-driven migration and invasion.

cell biology↗

bioRxiv2018

ERK1/2-dependent activation of FCHSD2 drives 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 reciprocally regulates CME in cancer cells and that the 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. Inhibition of several kinases selectively affected CME function in cancer cells. Among these, ERK1/2 inhibition selectively inhibited CME in cancer cells by decreasing the rate of 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. ERK1/2 phosphorylation activates FCHSD2 and regulates EGFR endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in non-small-cell lung cancer 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 cancer patient survival rate, suggesting that FCHSD2 negatively affects cancer progression. These findings provide new insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.\n\nSignificanceClathrin-mediated endocytosis (CME) determines the internalization of receptors and their downstream signaling. We discovered that CME is differentially regulated by specific signaling kinases in cancer cells. In particular, ERK1/2-mediated phosphorylation of the FCH/F-BAR and double SH3 domains-containing protein 2 (FCHSD2) regulates CME, and the trafficking and signaling activities of EGF receptors. This reciprocal interaction negatively regulates cancer proliferation and migration. The expression level of FCHSD2 is positively correlated with higher cancer patient survival rates. This study identifies signaling pathways that impinge on the endocytic machinery and reveals a molecular nexus for crosstalk between intracellular signaling and CME. Cancer cells specifically adapt this crosstalk as a determinant for tumor progression, which has implications for novel therapeutics against cancers.

cell biology↗

bioRxiv2018

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

Dynamin 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 dynamin-2 (Dyn2), but inactivated in non-neuronal cells via phosphorylation by GSK3{beta} 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 burst of assembly prior to CCV formation. Activation of Dyn1 by acute inhibition of GSK3{beta} 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 even when present at low, substoichiometric levels relative to Dyn2, and apparently without assembly into supramolecular collar-like structures. Under physiological conditions Dyn1 is activated downstream of EGF-receptor 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, isoformspecific regulatory role for Dyn1 in clathrin-mediated endocytosis.

cell biology↗

bioRxiv2017

A high-throughput assay to identify robust inhibitors of dynamin GTPase activity

Clathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamins GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamins stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamins basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several \"hits\" that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamins basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for robust dynamin inhibitors.

biochemistry↗