Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with wide ranges of human diseases, e.g. cancer and neurodegenerative diseases. Although enormous progress has been made on the core molecular machineries of autophagy, mechanisms of autophagy induction, autophagosome maturation, and autophagosomal-lysosomal fusion still remain elusive in mammalian cells. 　　The two pore channel 2 (TPC2) is a new member of the superfamily of voltage-gated Na+/Ca2+ channels located on lysosomes. Accumulating evidence indicates that nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca2+ mobilizing nucleotides, elicits Ca2+ release from lysosomes via TPC2 in many cell types. Herein, we first studied the role and mechanism of NAADP/TPC2/Ca2+ signaling in regulation of autophagy in mammalian cells. We found that overexpression TPC2 in Hela cells or mouse embryonic stem (ES) cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with NAADP-AM, a cell-permeant NAADP agonist, further induced the accumulation, whereas Ned-19, a NAADP antagonist, reversed it. Interestingly, inhibiting MTOR activity failed to increase TPC2-induced autophagosome accumulation, but ATG5 knockdown markedly blocked it. Importantly, overexpression of TPC2 alkalinized lysosomal pH, whereas lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that NAADP/TPC2/Ca2+ signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression. Development of agonists or antagonists of NAADP should provide a novel approach to specifically manipulate autophagy. 　　Along this line, a large number of small chemicals that modulate autophagy have actually been widely used to dissect this process and some of them, e.g. chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Yet most of the autophagy chemical modulators lack specificity, or potency, or both. Therefore we screened a panel of small chemicals that are commercially available and have been previously shown to affect vesicle trafficking or organelle morphology on autophagy regulation. We found that vacuolin-1, a cell permeable small molecule, potently and reversibly inhibited autophagosomal-lysosomal fusion in mammalian cells, thereby inducing the accumulation of autophagosomes. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Interestingly, treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated Rab5 GTPase activity. Expression of a dominant negative mutant of Rab5A or Rab5A knockdown significantly inhibited vacuolin-1 induced autophagosomal-lysosomal fusion blockage, whereas expression of a constitutive active form of Rab5A suppressed the lysosomal-autophagosomal fusion. Taken together, these data suggest that vacuolin-1 activates Rab5A to suppress the autophagosomal-lysosomal fusion, possibly via alkalizing lysosomal pH. Moreover, vacuolin-1 treatment alone showed little cell toxicity, but markedly suppressed the colony formation and migration of tumor cells in vitro. Therefore, vacuolin-1 and its analogues present a novel class of drug that can potently and reversibly modulate autophagy, and are potential drugs for treating autophagy related human diseases, e.g. cancer. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Errata sheet has been pasted onto reverse of back end paper. / Bibliography: leaves 116-145. / 145, [44] leaves, [24] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Apoptosis, or programmed cell death, is a fundamental process during both embryonic development and adult homeostasis. Aberrant apoptosis has been associated with a number of human diseases, which may arise either from a failure of cells to undergo apoptosis, or from excessive apoptosis. Understanding of the cellular components of the apoptotic machinery should contribute to the design of therapeutic agents which may be beneficial in the treatment of diseases which have disruptions in the apoptotic pathway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998

Errata sheet has been pasted onto reverse of back end paper. / Bibliography: leaves 116-145. / 145, [44] leaves, [24] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Apoptosis, or programmed cell death, is a fundamental process during both embryonic development and adult homeostasis. Aberrant apoptosis has been associated with a number of human diseases, which may arise either from a failure of cells to undergo apoptosis, or from excessive apoptosis. Understanding of the cellular components of the apoptotic machinery should contribute to the design of therapeutic agents which may be beneficial in the treatment of diseases which have disruptions in the apoptotic pathway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998

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