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.
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.
|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:|
|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|
|He, Kangmin; Marsland Iii, Robert; Upadhyayula, Srigokul et al. (2017) Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic. Nature 552:410-414|
|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|
|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|
|Chou, Yi-ying; Cuevas, Christian; Carocci, Margot et al. (2016) Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor. J Virol 90:4494-510|
|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|
|Wang, Huajin; Becuwe, Michel; Housden, Benjamin E et al. (2016) Seipin is required for converting nascent to mature lipid droplets. Elife 5:|
|Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun et al. (2015) Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology. Haematologica 100:439-51|
|Cocucci, Emanuele; Meldolesi, Jacopo (2015) Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol 25:364-72|
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