Understanding the role of transporters and ion channels in lysosomal homeostasis
Cellular activity is regulated based on a range of cues to maintain homeostasis. The lysosome is a key regulator of cellular activity as it acts as a signaling hub for metabolic regulation and is the main site of biomolecule degradation. Following the degradation of biomolecules, including proteins,...
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| Format: | Thesis |
| Language: | English |
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2024
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| _version_ | 1824458889214558208 |
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| author | Salphati, S |
| author2 | Newstead, S |
| author_facet | Newstead, S Salphati, S |
| author_sort | Salphati, S |
| collection | OXFORD |
| description | Cellular activity is regulated based on a range of cues to maintain homeostasis. The lysosome is a key regulator of cellular activity as it acts as a signaling hub for metabolic regulation and is the main site of biomolecule degradation. Following the degradation of biomolecules, including proteins, glycans, nucleic acids and lipids, nutrients are exported out of the lysosome by efflux transporters, which provide nutrients for cellular anabolic activity. Efflux transporters maintain lysosomal homeostasis by preventing the deleterious accumulation of nutrients within the lumen. Here, the mechanism of cystine transport at the lysosomal membrane by cystinosin is characterized using two-electrode voltage clamp. This work provides novel insights into the mechanisms that maintain nutrient homeostasis in lysosomes, and how they are disrupted in the lysosomal storage disorder cystinosis. Lysosomal homeostasis is also preserved by protein signaling, which regulates lysosome biogenesis, trafficking, fusion, and activity. A key regulator of cell signaling at the lysosomal membrane is the ion channel TRPML1. Numerous functions have been associated with TRPML1 activity, but its mechanisms of action remain poorly understood. This is in part due to the lack of specific tools to investigate its functions (modulators) and localization (antibodies). Nanobodies have previously been used as tools to investigate the mechanisms and localization of membrane proteins in cells. Here, high-affinity TRPML1 synthetic nanobodies (sybodies) were generated and characterized biophysically and structurally. A TRPML1-specific bivalent construct was engineered and applied to STochastic Optical Reconstruction Microscopy (STORM) imaging and immunoprecipitation of endogenous TRPML1. Future work will aim to use these binders and protocols to investigate the role of TRPML1 in neurodegeneration, which can result from the loss of lysosomal homeostasis. Moreover, the protocols described here will enable to engineer sybodies against additional lysosomal targets to investigate their role in lysosomal homeostasis. |
| first_indexed | 2025-02-19T04:33:03Z |
| format | Thesis |
| id | oxford-uuid:61a58b65-37a9-450b-a45d-c202dbd79a4a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:33:03Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:61a58b65-37a9-450b-a45d-c202dbd79a4a2025-01-20T10:21:38ZUnderstanding the role of transporters and ion channels in lysosomal homeostasisThesishttp://purl.org/coar/resource_type/c_db06uuid:61a58b65-37a9-450b-a45d-c202dbd79a4aEnglishHyrax Deposit2024Salphati, SNewstead, STucker, SCellular activity is regulated based on a range of cues to maintain homeostasis. The lysosome is a key regulator of cellular activity as it acts as a signaling hub for metabolic regulation and is the main site of biomolecule degradation. Following the degradation of biomolecules, including proteins, glycans, nucleic acids and lipids, nutrients are exported out of the lysosome by efflux transporters, which provide nutrients for cellular anabolic activity. Efflux transporters maintain lysosomal homeostasis by preventing the deleterious accumulation of nutrients within the lumen. Here, the mechanism of cystine transport at the lysosomal membrane by cystinosin is characterized using two-electrode voltage clamp. This work provides novel insights into the mechanisms that maintain nutrient homeostasis in lysosomes, and how they are disrupted in the lysosomal storage disorder cystinosis. Lysosomal homeostasis is also preserved by protein signaling, which regulates lysosome biogenesis, trafficking, fusion, and activity. A key regulator of cell signaling at the lysosomal membrane is the ion channel TRPML1. Numerous functions have been associated with TRPML1 activity, but its mechanisms of action remain poorly understood. This is in part due to the lack of specific tools to investigate its functions (modulators) and localization (antibodies). Nanobodies have previously been used as tools to investigate the mechanisms and localization of membrane proteins in cells. Here, high-affinity TRPML1 synthetic nanobodies (sybodies) were generated and characterized biophysically and structurally. A TRPML1-specific bivalent construct was engineered and applied to STochastic Optical Reconstruction Microscopy (STORM) imaging and immunoprecipitation of endogenous TRPML1. Future work will aim to use these binders and protocols to investigate the role of TRPML1 in neurodegeneration, which can result from the loss of lysosomal homeostasis. Moreover, the protocols described here will enable to engineer sybodies against additional lysosomal targets to investigate their role in lysosomal homeostasis. |
| spellingShingle | Salphati, S Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title | Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title_full | Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title_fullStr | Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title_full_unstemmed | Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title_short | Understanding the role of transporters and ion channels in lysosomal homeostasis |
| title_sort | understanding the role of transporters and ion channels in lysosomal homeostasis |
| work_keys_str_mv | AT salphatis understandingtheroleoftransportersandionchannelsinlysosomalhomeostasis |