Reorganization of cellular membranes and transport of components from one lipid-bilayer bounded compartment to another underlie much of the internal structure of a cell. Clathrin is the principal molecular scaffold for a number of such processes -- most notably, receptor-mediated endocytosis of ligands such as transferrin, LDL, growth factors, and hormones. Viruses and other pathogens and pathogenic toxins usurp this pathway to enter cells. Direct observation of the biochemistry of clathrin-dependent membrane traffic in living cells is now possible, through a combination of genome editing and single-fluorophore sensitivity imaging. The combination, which amounts to in vivo, single-molecule biochemistry, resolves ambiguities about essential components and the time points at which they function, particularly when many of the steps have a stochastic rather than fully deterministic character. Our work in the previous grant period on the molecular mechanism of coated-pit initiation illustrates the value of single-fluorophore-sensitivity imaging approaches. I the research described in this proposal, we will analyze the molecular mechanisms of transitional checkpoints in coat assembly, develop and apply probes for the roles of specific phosphoinositide lipids in regulating clathrin- based membrane traffic, and extend our imaging biochemistry to the 3D context of multicellular assemblies and living tissues. We will capitalize on a transformative new imaging technology, lattice light- sheet microscopy (LLSM), which enables rapid, high-resolution, high-sensitivity 3D visualization of whole cells and multicellular specimens, including living tissues.

Public Health Relevance

We will apply transformative new technologies to broaden and deepen understanding of the process that allows cells to import molecules from their surroundings (including pathogens and toxins that usurp the pathway), to regulate the presence of receptors on their surfaces, and to transmit signals between cells in tissues.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Maas, Stefan
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Boston Children's Hospital
United States
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Coulter, Michael E; Dorobantu, Cristina M; Lodewijk, Gerrald A et al. (2018) The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles. Cell Rep 24:973-986.e8
Swinburne, Ian A; Mosaliganti, Kishore R; Upadhyayula, Srigokul et al. (2018) Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure. Elife 7:
Liu, Tsung-Li; Upadhyayula, Srigokul; Milkie, Daniel E et al. (2018) Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms. Science 360:
Schuler, Max-Hinderk; Lewandowska, Agnieszka; Caprio, Giuseppe Di et al. (2017) Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration. Mol Biol Cell 28:2159-2169
Adell, Manuel Alonso Y; Migliano, Simona M; Upadhyayula, Srigokul et al. (2017) Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding. Elife 6:
He, Kangmin; Marsland III, Robert; Upadhyayula, Srigokul et al. (2017) Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic. Nature 552:410-414
Chou, Yi-Ying; Cuevas, Christian; Carocci, Margot et al. (2016) Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor. J Virol 90:4494-4510
Hu, Yu; Kim, Ji Hyung; He, Kangmin et al. (2016) Scramblase TMEM16F terminates T cell receptor signaling to restrict T cell exhaustion. J Exp Med 213:2759-2772
Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël et al. (2016) Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy. Mol Biol Cell 27:3418-3435
Reeves, Patrick M; Kang, Yuan-Lin; Kirchhausen, Tom (2016) Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway. Traffic 17:40-52

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