Hinze, Claudia; (2018) Changes in endocytosis and trafficking during proliferative quiescence. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A significant proportion of cells in the adult human body, including naïve lymphocytes, hepatocytes, stem cells and cancer stem cells, reside in an actively maintained state of proliferative quiescence (G0). Quiescent cells exit the cell cycle under certain conditions and resume proliferation upon appropriate stimuli, which distinguishes them from senescent or terminally differentiated cells. Little is known about how molecular processes, such as endocytosis, are regulated upon cell cycle exit. The aim of this study was to measure endocytic pathways during quiescence and study how their regulation contributes to quiescence maintenance. A quiescence induction protocol was optimised for hTERT-immortalised RPE1 (retina pigmented epithelial) cells to compare endocytosis between quiescent and proliferating RPE1 cells. A SILAC mass spectrometry screen comparing proteome and phosphoproteome between G0 and G1 RPE1 cells revealed changes in total protein levels and phosphorylation of endocytic proteins during G0. Confocal microscopy, Western blotting, flow cytometry and high throughput imaging techniques were used to measure clathrin-mediated (CME) and -independent endocytosis in quiescent and continuously proliferating RPE1 cells. Total levels of core proteins of the clathrin machinery were increased during G0, but uptake of the classical CME cargoes transferrin, EGF and LDL were decreased. CME activity during quiescence is cargo-specific, as could be shown for elevated Lamp1 endocytosis. Endocytosis of clathrin-independent cargoes such as oxidised LDL and Cholera toxin was highly active in quiescent cells, as was uptake of the acropinocytosis cargoes dextran and BSA. Elevated BSA uptake, however, did not promote mTORC1-mediated survival in a nutrient-(amino acid-) deprived environment. Moreover, BSA endocytosis was mediated by AP2. Finally, quiescence survival signalling via integrins was found to be dependent on endocytosis and recycling. Together, this study identified differentially regulated endocytic pathways and suggestd a role for integrin trafficking to maintain proliferative quiescence.

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