Clathrin-mediated endocytosis (CME) is the major pathway for internalization from, and remodeling of the plasma membrane (PM) in mammalian cells. Thus, CME plays a fundamental role in all aspects of cell physiology, including nutrient uptake, signal transduction, cell motility, adhesion, polarity and differentiation. Consequently, defects in CME impinge on many human diseases, including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent CCPs. Subsequent steps including CCP stabilization, maturation and their scission from the PM to release clathrin coated vesicles are orchestrated by a myriad of endocytic accessory proteins (EAPs). Under the auspices of this grant, we have built an interdisciplinary team of biochemists, cell and molecular biologists, microscopists, engineers and computational biologists whose long- term goal is to define the mechanisms that regulate CME. Over the past 8 years, we have developed accurate and highly sensitive particle detection and tracking software and algorithms to quantitatively measure multiple orthogonal parameters relating to the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence microscopy. We identified 3 kinetically-distinct subpopulations of CCPs, two short-lived abortive populations and longer-lived productive CCPs, as well as factors that differentially affect CCP initiation, stabilization and maturation. These data led us to propose that CCP maturation is gated during the first ~30s after initiation by an "endocytic check-point" that is, in part, regulated by the GTPase dynamin. Early recruitment of AP2-interacting EAPs to CCPs is also required for efficient curvature generation and to regulate multiple stages of the maturation process. The overarching goal of this proposal is to test this checkpoint hypothesis by identifying the determinants (i.e. individual or sets of EAPs) that function as potential sensors or effectors of the checkpoint, as well as those required for efficient CCP stabilization and maturation. To accomplish these goals, we propose three Specific Aims: 1) To measure the effect of individual and pairwise knockdown of EAPs on stages of CME through quantitative multi-parametric analyses of CCP dynamics;2) To develop a molecularly explicit, mathematical model of the multi-step CCP maturation program and calibrate model parameters against measured CCP lifetime distributions in siRNA-treated cells, and 3) To, directly test, under minimally perturbing conditions, the functional assignments of EAPs predicted from the studies in Aim 1 and 2, and define the functional hierarchy of EAPs in regulating CCP maturation and progression beyond the endocytic checkpoint. These studies will provide unprecedented insights into the function of individual EAPs and their role in regulating key early stages of CCP stabilization of maturation. While most studies have focused on readily detectable early (CCP initiation) or late (CCV budding) events, we focus here on the less well understood stages of CCP stabilization and maturation that are central to the regulation of CME.

Public Health Relevance

The goal of this proposal is to define the molecular mechanisms that regulate clathrin-mediated endocytosis (CME). CME is the major pathway for internalization of surface receptors and transporters and controls the composition of the plasma membrane, which is the platform through which cells communicate with each other and with their environment. Thus, regulation of CME is essential for many aspects of cell and organismal physiology and homeostasis and defects in the regulation of CME are linked to neurological disorders, hypercholesterolemia, diabetes and cancer. Identifying key factors that regulate CME could provide new therapeutic strategies to shift the balance of cell surface receptors as a means of altering signal transduction in disease-related cell pathologies.

National Institute of Health (NIH)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Ainsztein, Alexandra M
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University of Texas Sw Medical Center Dallas
Anatomy/Cell Biology
Schools of Medicine
United States
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Aguet, Francois; Antonescu, Costin N; Mettlen, Marcel et al. (2013) Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint. Dev Cell 26:279-91
Danuser, Gaudenz; Allard, Jun; Mogilner, Alex (2013) Mathematical modeling of eukaryotic cell migration: insights beyond experiments. Annu Rev Cell Dev Biol 29:501-28
Sharma, Sonia; Quintana, Ariel; Findlay, Gregory M et al. (2013) An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry. Nature 499:238-42
Dittrich, Christian; Burckhardt, Christoph J; Danuser, Gaudenz (2012) Delivery of membrane impermeable cargo into CHO cells by peptide nanoparticles targeted by a protein corona. Biomaterials 33:2746-53
Antonescu, Costin N; Aguet, Francois; Danuser, Gaudenz et al. (2011) Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size. Mol Biol Cell 22:2588-600
Bocking, Till; Aguet, Francois; Harrison, Stephen C et al. (2011) Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18:295-301
Nunez, Daniel; Antonescu, Costin; Mettlen, Marcel et al. (2011) Hotspots organize clathrin-mediated endocytosis by efficient recruitment and retention of nucleating resources. Traffic 12:1868-78
Loerke, Dinah; Mettlen, Marcel; Schmid, Sandra L et al. (2011) Measuring the hierarchy of molecular events during clathrin-mediated endocytosis. Traffic 12:815-25
Mettlen, Marcel; Loerke, Dinah; Yarar, Defne et al. (2010) Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis. J Cell Biol 188:919-33
Antonescu, Costin N; Danuser, Gaudenz; Schmid, Sandra L (2010) Phosphatidic acid plays a regulatory role in clathrin-mediated endocytosis. Mol Biol Cell 21:2944-52

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