Mechanisms Regulating Clathrin-Mediated Endocytosis

Summary

Principal Investigator: SANDRA L contact SCHMID
Affiliation: The Scripps Research Institute
Country: USA
Abstract: DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) occurs via the assembly of clathrin-coated pits (CCPs), specialized microdomains at the plasma membrane that undergo progressive maturation and eventually invaginate and pinch-off to form sealed clathrin-coated vesicles (CCV) carrying their cargo (receptor-ligand complexes) into the cell. In the past funding period we probed by quantitative live cell imaging the dynamics of CCP formation and maturation and tested the role(s) of actin assembly in CME. We developed image analysis software to track in an unbiased way every clathrin-coated structure (CCS) visible by total internal reflection fluorescence microscopy relative to actin cortex dynamics. Focusing first on the heterogeneous lifetimes of CCPs we identified by decomposition of the lifetime histogram two shorter-lived and one longer-lived sub-population, each by itself exhibiting a wide spectrum of lifetimes. Systematic RNAi and decomposition of the shifted lifetime distributions revealed endocytic accessory factors that affect either the relative contribution or the lifetime of one of the three subpopulations. From these data we derived a working model of the hierarchy of molecular events during CME, including the requirements for actin filaments to build a scaffold during the initiation of CCPs and to depolymerize and differentially repolymerize in a pit-size dependent fashion during internalization. Intriguingly, our data posit the existence of a restriction/checkpoint that ~20-30 s after initiation gates CCP progression to a productive CCS versus abortion. Our data suggests that checkpoint ris regulated by the GTPase dynamin and is sensitive to cargo concentration and CCP composition. Armed with this initial success in image-based analyses of CME, we propose in the next funding period to test the hypotheses that i) through interaction with early accessory factors of the endocytic machinery, dynamin serves to integrate multiple physical and chemical cues and to monitor the fidelity of CCP assembly;ii) the concentration and stoichiometry of cargo-specific adaptors are essential determinants of the efficiency of CCP maturation;and iii) that the molecular composition of CCPs is an essential determinant of both the efficiency and rate of CCP maturation. To conduct these studies we will develop new, highly sensitive and quantitative imaging assays to determine the molecular composition of individual CCPs. Next, we will devise deterministic and probabilistic mathematical models to integrate the data and to make molecularly explicit predictions of the interactions between endocytic accessory factors and their functions in the CME program. Our primary approach is to exploit the intrinsic heterogeneity in the wildtype composition and dynamics of CCPs as a source of information for modeling, avoiding the notorious distortions associated with molecular intervention studies. To accomplish this we will produce cell models, imaging protocols and data analysis tools that will raise the sensitivity of our experiments to a level where model predictions can also be validated by very mild perturbation experiments that do not affect the overall homeostasis of CME. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop quantitative imaging assays and mathematical models to decipher the regulation of the early stages of clathrin-mediate endocytosis at the cell surface. Understanding the mechanisms of initiation of endocytosis is critical to augmenting current therapeutic strategies that target the balance of cell surface receptors as a means of altering signal transduction in disease-related cell pathologies. Understanding the initiation of endocytosis is also critical to fight the proliferation of bacteria and viruses, which hijack the endocytic machinery to enter their host cells.
Funding Period: ----------------2006 - ---------------2014-
more information: NIH RePORT

Top Publications

  1. pmc Robust single-particle tracking in live-cell time-lapse sequences
    Khuloud Jaqaman
    Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, California 92037, USA
    Nat Methods 5:695-702. 2008
  2. pmc Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint
    François Aguet
    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
    Dev Cell 26:279-91. 2013
  3. pmc An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry
    Sonia Sharma
    La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA
    Nature 499:238-42. 2013
  4. pmc The first five seconds in the life of a clathrin-coated pit
    Emanuele Cocucci
    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
    Cell 150:495-507. 2012
  5. pmc Mathematical modeling of eukaryotic cell migration: insights beyond experiments
    Gaudenz Danuser
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 email
    Annu Rev Cell Dev Biol 29:501-28. 2013
  6. pmc Local clustering of transferrin receptors promotes clathrin-coated pit initiation
    Allen P Liu
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 191:1381-93. 2010
  7. pmc Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis
    Marcel Mettlen
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 188:919-33. 2010
  8. pmc Cargo and dynamin regulate clathrin-coated pit maturation
    Dinah Loerke
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
    PLoS Biol 7:e57. 2009
  9. pmc Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits
    Marcel Mettlen
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Mol Biol Cell 20:3251-60. 2009
  10. pmc Global and local regulation of clathrin-coated pit dynamics detected on patterned substrates
    Allen P Liu
    Department of Cell Biology, Scripps Research Institute, La Jolla, California, USA
    Biophys J 97:1038-47. 2009

Scientific Experts

  • TOMAS L KIRCHHAUSEN
  • S L Schmid
  • Dinah Loerke
  • Christian Dittrich
  • Gaudenz Danuser
  • Marcel Mettlen
  • François Aguet
  • Costin N Antonescu
  • Allen P Liu
  • Sonia Sharma
  • Emanuele Cocucci
  • Till Böcking
  • Daniel Núñez
  • Thomas J Pucadyil
  • Khuloud Jaqaman
  • Ariel Quintana
  • Beate Baust
  • Jun Allard
  • Patrick G Hogan
  • Alex Mogilner
  • Mohit Jain
  • Anjana Rao
  • Gregory M Findlay
  • Roland Nilsson
  • Steeve Boulant
  • Costin Antonescu
  • Stephen C Harrison
  • Allen Liu
  • Defne Yarar
  • Miriam Stoeber
  • Thomas Pucadyil
  • Rajesh Ramachandran
  • Hirotaka Kuwata
  • Sergio Grinstein

Detail Information

Publications18

  1. pmc Robust single-particle tracking in live-cell time-lapse sequences
    Khuloud Jaqaman
    Department of Cell Biology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, California 92037, USA
    Nat Methods 5:695-702. 2008
    ..Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane...
  2. pmc Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint
    François Aguet
    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
    Dev Cell 26:279-91. 2013
    ..We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME...
  3. pmc An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry
    Sonia Sharma
    La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA
    Nature 499:238-42. 2013
    ..Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes. ..
  4. pmc The first five seconds in the life of a clathrin-coated pit
    Emanuele Cocucci
    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
    Cell 150:495-507. 2012
    ..This objective picture of coated pit initiation also shows that methods outlined here will be broadly useful for studies of dynamic assemblies in living cells...
  5. pmc Mathematical modeling of eukaryotic cell migration: insights beyond experiments
    Gaudenz Danuser
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 email
    Annu Rev Cell Dev Biol 29:501-28. 2013
    ....
  6. pmc Local clustering of transferrin receptors promotes clathrin-coated pit initiation
    Allen P Liu
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 191:1381-93. 2010
    ..Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs...
  7. pmc Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis
    Marcel Mettlen
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    J Cell Biol 188:919-33. 2010
    ..Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs...
  8. pmc Cargo and dynamin regulate clathrin-coated pit maturation
    Dinah Loerke
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
    PLoS Biol 7:e57. 2009
    ..From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin...
  9. pmc Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits
    Marcel Mettlen
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Mol Biol Cell 20:3251-60. 2009
    ....
  10. pmc Global and local regulation of clathrin-coated pit dynamics detected on patterned substrates
    Allen P Liu
    Department of Cell Biology, Scripps Research Institute, La Jolla, California, USA
    Biophys J 97:1038-47. 2009
    ..Although we cannot rule out the possibility that other factors might locally affect CCP dynamics at Fn islands, our data suggest that localized modulation in cortical tension may spatially regulate clathrin-mediated endocytosis...
  11. pmc Delivery of membrane impermeable cargo into CHO cells by peptide nanoparticles targeted by a protein corona
    Christian Dittrich
    Dept of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
    Biomaterials 33:2746-53. 2012
    ..These data indicate corona-mediated delivery of membrane impermeable cargo in vitro by a particulate delivery system entirely composed of amino acids...
  12. pmc Hotspots organize clathrin-mediated endocytosis by efficient recruitment and retention of nucleating resources
    Daniel Núñez
    Department of Cell Biology, Harvard Medical School, Boston, MA, USA
    Traffic 12:1868-78. 2011
    ....
  13. pmc Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size
    Costin N Antonescu
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Mol Biol Cell 22:2588-600. 2011
    ..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...
  14. pmc Measuring the hierarchy of molecular events during clathrin-mediated endocytosis
    Dinah Loerke
    Department of Physics and Astronomy, University of Denver, Denver, CO 80208, USA
    Traffic 12:815-25. 2011
    ..This method provides a new means to identify other such hierarchies during CCP maturation...
  15. pmc Conserved functions of membrane active GTPases in coated vesicle formation
    Thomas J Pucadyil
    Department of Cell Biology, The Scripps Research Institute TSRI, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
    Science 325:1217-20. 2009
    ....
  16. pmc Dissecting dynamin's role in clathrin-mediated endocytosis
    Marcel Mettlen
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
    Biochem Soc Trans 37:1022-6. 2009
    ....
  17. pmc Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis
    Costin N Antonescu
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA
    Mol Biol Cell 21:2944-52. 2010
    ..These findings demonstrate that PA plays a regulatory rather than obligatory role in CME and differentially regulates ligand-stimulated CME of EGFR...
  18. pmc Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating
    Till Böcking
    Immune Disease Institute, Harvard Medical School, Boston, MA, USA
    Nat Struct Mol Biol 18:295-301. 2011
    ..Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes...