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.

[1]  T. Kirchhausen,et al.  Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic , 2020, The Journal of cell biology.

[2]  P. De Camilli,et al.  Dynamic instability of clathrin assembly provides proofreading control for endocytosis , 2019, The Journal of cell biology.

[3]  Y. Ouyang,et al.  AAGAB Controls AP2 Adaptor Assembly in Clathrin-Mediated Endocytosis. , 2019, Developmental cell.

[4]  Marcel Mettlen,et al.  Regulation of Clathrin-Mediated Endocytosis. , 2018, Annual review of biochemistry.

[5]  S. Schmid,et al.  A noncanonical role for dynamin-1 in regulating early stages of clathrin-mediated endocytosis in non-neuronal cells , 2018, PLoS biology.

[6]  William J. Godinez,et al.  Clathrin-adaptor ratio and membrane tension regulate the flat-to-curved transition of the clathrin coat during endocytosis , 2018, Nature Communications.

[7]  M. Kaksonen,et al.  Mechanisms of clathrin-mediated endocytosis , 2018, Nature Reviews Molecular Cell Biology.

[8]  V. Haucke,et al.  A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch. , 2017, Developmental cell.

[9]  Marcel Mettlen,et al.  Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature , 2017, The Journal of cell biology.

[10]  G. Hollopeter,et al.  NECAPs are negative regulators of the AP2 clathrin adaptor complex , 2017, bioRxiv.

[11]  E. Johnston-Halperin,et al.  Membrane mechanics govern spatiotemporal heterogeneity of endocytic clathrin coat dynamics , 2017, Molecular biology of the cell.

[12]  S. Schmid,et al.  Endocytosis, Metastasis and Beyond: Multiple Facets of SNX9. , 2017, Trends in cell biology.

[13]  Marcel Mettlen,et al.  Crosstalk between CLCb/Dyn1-Mediated Adaptive Clathrin-Mediated Endocytosis and Epidermal Growth Factor Receptor Signaling Increases Metastasis. , 2017, Developmental cell.

[14]  Stephanie J. Spielman,et al.  Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2 , 2017, The Journal of cell biology.

[15]  A. Audhya,et al.  Eps15 membrane-binding and -bending activity acts redundantly with Fcho1 during clathrin-mediated endocytosis , 2016, Molecular biology of the cell.

[16]  Simon C Watkins,et al.  Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding , 2016, Developmental cell.

[17]  Erik S. Welf,et al.  SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases , 2016, Molecular biology of the cell.

[18]  D. Drubin,et al.  Machine-Learning-Based Analysis in Genome-Edited Cells Reveals the Efficiency of Clathrin-Mediated Endocytosis. , 2015, Cell reports.

[19]  F. Aguet,et al.  Crosstalk between Akt/GSK3β signaling and dynamin‐1 regulates clathrin‐mediated endocytosis , 2015, The EMBO journal.

[20]  J. Briggs,et al.  Endocytic sites mature by continuous bending and remodeling of the clathrin coat , 2015, Science.

[21]  J. Graff,et al.  Drosophila glucome screening identifies Ck1alpha as a regulator of mammalian glucose metabolism , 2015, Nature Communications.

[22]  Sharon E. Miller,et al.  CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature , 2015, Developmental cell.

[23]  Marcel Mettlen,et al.  Imaging and modeling the dynamics of clathrin-mediated endocytosis. , 2014, Cold Spring Harbor perspectives in biology.

[24]  T. Kirchhausen,et al.  Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit , 2014, Molecular biology of the cell.

[25]  Simon C Watkins,et al.  A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing , 2014, eLife.

[26]  D. Drubin,et al.  Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis , 2014, The Journal of cell biology.

[27]  David Owen,et al.  Molecular structure, function, and dynamics of clathrin-mediated membrane traffic. , 2014, Cold Spring Harbor perspectives in biology.

[28]  Volker Haucke,et al.  Spatiotemporal control of endocytosis , 2014 .

[29]  E. Jorgensen,et al.  NECAP 1 Regulates AP-2 Interactions to Control Vesicle Size, Number, and Cargo During Clathrin-Mediated Endocytosis , 2013, PLoS biology.

[30]  Sandra L Schmid,et al.  Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint. , 2013, Developmental cell.

[31]  Frank Noé,et al.  Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate , 2013, Nature.

[32]  Michael T. McManus,et al.  A Systematic Mammalian Genetic Interaction Map Reveals Pathways Underlying Ricin Susceptibility , 2013, Cell.

[33]  L. Collinson,et al.  A Human Genome-wide Screen for Regulators of Clathrin-coated Vesicle Formation Reveals an Unexpected Role for the V-ATPase , 2012, Nature Cell Biology.

[34]  F. Aguet,et al.  The First Five Seconds in the Life of a Clathrin-Coated Pit , 2012, Cell.

[35]  Qingli Xiao,et al.  Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis* , 2012, The Journal of Biological Chemistry.

[36]  Marcus J. Taylor,et al.  A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis , 2012, PLoS biology.

[37]  Adi Pick,et al.  Membrane Fission Is Promoted by Insertion of Amphipathic Helices and Is Restricted by Crescent BAR Domains , 2012, Cell.

[38]  Sandra L Schmid,et al.  Measuring the Hierarchy of Molecular Events During Clathrin‐Mediated Endocytosis , 2011, Traffic.

[39]  D. Perrais,et al.  A High Precision Survey of the Molecular Dynamics of Mammalian Clathrin-Mediated Endocytosis , 2011, Microscopy and Microanalysis.

[40]  F. Aguet,et al.  Local clustering of transferrin receptors promotes clathrin-coated pit initiation , 2010, The Journal of cell biology.

[41]  P. Evans,et al.  A Large-Scale Conformational Change Couples Membrane Recruitment to Cargo Binding in the AP2 Clathrin Adaptor Complex , 2010, Cell.

[42]  S. Keeney,et al.  References and Notes Supporting Online Material Materials and Methods Figs. S1 to S5 Tables S1 and S2 References Movie S1 Fcho Proteins Are Nucleators of Clathrin-mediated Endocytosis , 2022 .

[43]  Y. Kalaidzidis,et al.  Systems survey of endocytosis by multiparametric image analysis , 2010, Nature.

[44]  Nick C Fox,et al.  Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease, and shows evidence for additional susceptibility genes , 2009, Nature Genetics.

[45]  D. Loerke,et al.  Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits. , 2009, Molecular biology of the cell.

[46]  Sandra L Schmid,et al.  Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation , 2009, PLoS biology.

[47]  Sharon E. Miller,et al.  A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex , 2008, Nature.

[48]  K. Jaqaman,et al.  Robust single particle tracking in live cell time-lapse sequences , 2008, Nature Methods.

[49]  T. Kirchhausen,et al.  Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane. , 2008, Biophysical journal.

[50]  A. Griffin,et al.  A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating , 2006, Proceedings of the National Academy of Sciences.

[51]  Simon C Watkins,et al.  Epsin 1 is a Polyubiquitin‐Selective Clathrin‐Associated Sorting Protein , 2006, Traffic.

[52]  D. Loerke,et al.  Differential Control of Clathrin Subunit Dynamics Measured with EW‐FRAP Microscopy , 2005, Traffic.

[53]  U. Seifert.  Entropy production along a stochastic trajectory and an integral fluctuation theorem. , 2005, Physical review letters.

[54]  P. Evans,et al.  Adaptors for clathrin coats: structure and function. , 2004, Annual review of cell and developmental biology.

[55]  K. Chandran,et al.  Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits , 2004, Cell.

[56]  W. Marshall,et al.  Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference*[boxs] , 2004, Journal of Biological Chemistry.

[57]  Sandra L. Schmid,et al.  Regulated portals of entry into the cell , 2003, Nature.

[58]  P. Evans,et al.  Molecular Architecture and Functional Model of the Endocytic AP2 Complex , 2002, Cell.

[59]  E. Eisenberg,et al.  Clathrin exchange during clathrin-mediated endocytosis , 2001, The Journal of cell biology.

[60]  Francesca Santini,et al.  Spatial control of coated-pit dynamics in living cells , 1999, Nature Cell Biology.

[61]  Robert Pepperell,et al.  What is a Human? , 1994, Intell. Tutoring Media.

[62]  S. Schmid,et al.  Stage-specific assays for coated pit formation and coated vesicle budding in vitro , 1991, The Journal of cell biology.

[63]  Trupti M. Kodinariya,et al.  Review on determining number of Cluster in K-Means Clustering , 2013 .

[64]  Nick C Fox,et al.  Letter abstract - Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's Disease , 2009 .