除了將溶酶體視為降解過程的最後胞器，溶酶體逐漸被認為參與控制細胞功能的上游路徑。因此，溶酶體的動態穩定應該被嚴格的調控，已配合代謝的需求以及維護溶酶體參與的細胞功能調控。這裡我們使用光調控產生粒線體自噬需求，以降解基質影響溶酶體的動態穩定，並定量觀察在不同程度的降解壓力下，溶酶體如何調控其生合成與動態平衡。我們觀察到，在粒線體自噬降解需求下，TFEB轉錄因子調控的溶酶體相關基因有正向活化趨勢，並且基因活化幅度與降解需求有正相關性。細胞藉由調控mTOR的活性抑制，得以產生這種壓力與反應的定量協調。進一步的研究證實粒線體自噬造成的mTOR活性抑制是透過在autolysosome胞器上聚集mTOR活性調節分子，DEPDC5和FLCN。Autolysosome 是一種溶酶體與自噬體融合後的混和胞器。此外，藉由STX17基因靜默進而阻斷自噬體與溶酶體的融合，足以使TFEB基因活化反應與壓力脫鉤。這些結果顯示兩種胞器中，溶酶體和autolysosome在mTOR活性調控和TFEB基因活化的功能上有不同之處。溶酶體透過與自噬體的融合，得以定量上感測降解壓力的幅度並且微調TFEB基因活化以維護自我調節的動態平衡。

Except of the simplified view of lysosomes as the final compartments of degradation process, lysosomes are increasingly regarded as upstream organelles in the control of cell functions. Therefore, lysosome homeostasis should be tightly regulated to match the catabolic needs as well as to maintain lysosomal pathways. Here we use light-induced mitophagy substrates to disturb lysosome homeostatsis and reveal how lysosome biogenesis responses quantitatively to different levels of degradation stress. We observed that TFEB-mediated lysosomal genes activation is upregulated in dose-dependent manner upon variants mitophagy degradation stresses. This stress-response coordination is quantitatively modulated by mTOR inactivation. We further show that mitophagy-dependent mTOR inactivation is mediated by spatially recruiting mTOR activity regulators, DEPDC5 and FLCN, on autolysosome, a hybrid organelle of autophagosome and lysosome. Also, blocking autophagosome-lysosome fusion, by knocking down STX17, makes TFEB activation response decoupling from stress. These results suggest lysosome and autolysosome are functionally different in respect of mTOR activity and TFEB activation. Through fusing with autophagosomes, lysosomes sense degradation stress and decode into fine-tuning TFEB activation in the maintenance of self-regulated homeostasis.

1 Review of TFEB 9
1.1 TFEB related gene activation 9
1.2 TFEB neurodegenerative disease and development 11
1.3 TFEB regulation mechanism links to mTOR and lysosome 14
1.4 TFEB activated by different stresses which affect mTOR or lysosome 17
1.5 TFEB upregulated by itself and other transcription factor 19
2 Introduction 21
3 Results 23
3.1 TFEB-regulated genes activation coupling with degradation stress 23
3.2 Dephosphorylation on mTOR-responsive residue S211 is required for TFEB translocation under mitophagy degradation stress 26
3.3 GAP of Rag GTPase involving in mitophagy-mediated mTOR inhibition 28
3.4 Blocking autophagosome lysosome fusion inhibits mitophagy-dependent TFEB activation 31
4 Discussion 33
4.1 Photoinduced mitophagy 34
4.2 mTOR activity and mTOR substrate specificity 36
4.3 Rag GTPase regulated mTOR activity 39
4.4 Fusion dependent and hybrid organelle autophagolysosome 40
4.5 Lysosome homeostasis and stress response 42
5 Materials and Methods 45
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