Wednesday, April 27
Biblioteca - Sala Multiusos | 09:00 - 10:30
David Dubrin
Department of Molecular and Cell Biology, University of California, Berkeley, USA
David Dubrin, José Moura, Vitor Santos, Jaime Mota, Paula Videira, Pedro Viana Batista, Sergio Filipe, Ilda Sanches, Isabel Sá Nogueira.
Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Two recent advances offer great promise for accelerating progress in cell biology research and increasing the insights that can be gained. One is the development of genome editing technology. The other is the wide-spread availability of human stem cells that can be differentiated into cells of different fates.
Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyze endocytic dynamics in human cells in which endogenous protein stoichiometry is preserved, we are targeting zinc finger nucleases, TALENs and CRISPR/Cas9 to endocytic protein genomic loci and have generated numerous stable cell lines expressing combinations of fluorescent protein fusions from these genomic loci. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics. In the last few years, we have begun to edit human stem cells rather than tissue culture cells. These cells have normal ploidy, they can be differentiated into many different cell types isogenically, and they can be used to form tissue-like organoids for the study of cellular processes in the tissue context. CME dynamics are vastly different between fibroblasts, stem cells and neural progenitor cells.
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