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Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN ; Międzynarodowy Instytut Biologii Molekularnej i Komórkowej
Contributor:Miączyńska, Marta : Supervisor ; Cendrowski, Jarosław : Assistant supervisor
Publisher:Nencki Institute of Experimental Biology PAS
Place of publishing: Date issued/created: Description:131 pages : illustrations ; 30 cm ; Bibliography ; Summary in Polish
Degree grantor:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Type of object: Subject and Keywords:ESCRT ; Lysosome ; mTORC1 ; TFEB ; TFE3
Abstract:Endocytosis is a process of internalizing molecules from the extracellular milieu or the cell surface and delivering them to membrane-bound organelles called endosomes, which facilitate further transport of internalized cargoes. Proteins present on endosomal membranes are recognized by the endosomal sorting complexes required for transport (ESCRT), which consist of ESCRT-0, -I, -II and -III. ESCRT mediate remodeling of the limiting membrane of endosomes and formation of intraluminal vesicles (ILVs) inside endosomes. The content of ILVs can be secreted outside the cell or transported via the endolysosomal pathway to lysosomes for degradation. In addition, lysosomes regulate Ca2+-dependent signaling and constitute platforms to sense nutrient availability.Despite a well-characterized function of ESCRT-I in regulating endosomal size and sorting, its involvement in maintaining lysosomal homeostasis remains poorly investigated. The general aim of this thesis was to characterize the role of ESCRT-I in maintaining lysosomal homeostasis and investigate the consequences of ESCRT-I depletion for lysosomal function and lysosome-related signaling.First, lysosomal morphology was characterized in colorectal cancer cell lines, RKO and DLD-1, upon siRNA-mediated depletion of ESCRT-I components, namely Tsg101 or Vps28. Quantitative microscopic analysis of lysosomal markers revealed that lack of ESCRT-I led to enlargement of lysosomes but did not impair lysosomal integrity, maintenance of acidic pH or content of degradative enzymes. The increased lysosomal size was likely due to an impaired degradation of resident membrane proteins that was observed in cells lacking ESCRT-I. This included MCOLN1, a lysosomal Ca2+ channel, whose lysosomal degradation was studied using a GFP-MCOLN1-expressing reporter cell line.To verify whether the lack of ESCRT-I induced transcriptional responses characteristic for altered lysosomal function, RNA sequencing analysis was performed. It revealed that depletion of ESCRT-I upregulated expression of genes related to autophagy and/or lysosomal biogenesis. Activation of transcription factors from the MiT-TFE family, namely TFEB and TFE3, predicted to be responsible for induced expression of these genes, was confirmed in nuclear fractions of ESCRT-I-depleted cells.Next, a mechanism involved in the activation of MiT-TFE signaling upon ESCRT-I depletion was investigated. Quantitative analysis of microscopic images revealed that in cells lacking ESCRT-I, activation of TFEB and TFE3 required Ca2+-dependent signaling and mTORC1 inhibition, but was not due to calcineurin-dependent dephosphorylation of these transcription factors. Moreover, biochemical analyses indicated that the lack of ESCRT-I inhibited mammalian target of rapamycin complex 1 (mTORC1) kinase activity specific towards TFEB and TFE3 but it did not affect canonical mTORC1 substrates. Therefore, it was verified whether the MiT-TFE activation upon ESCRT-I depletion occurred due to the reduced activity of the Rag GTPase complex, known to control the TFEB- and TFE3-specific lysosomal mTORC1 signaling. Overexpression of constitutive active RagC mutant prevented nuclear translocation of TFEB and TFE3 in Tsg101-depleted cells. Hence, the activation of MiT-TFE factors in cells lacking ESCRT-I occurred due to the inhibition of Rag GTPase–dependent mTORC1 pathway.The results presented in this thesis characterize new roles of ESCRT-I in the turnover of lysosomal membrane proteins and maintaining lysosome-related Rag GTPase-dependent, non-canonical mTORC1 signaling. Lack of ESCRT-I leads to a homeostatic response, involving inhibition of the non-canonical mTORC1 signaling and, as a consequence, activation TFEB and TFE3 factors, in an attempt to counteract lysosomal nutrient starvation
Resource type: Detailed Resource Type: Source: Language: Language of abstract: Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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