Elucidating the mechanism of mRNA transport using novel imaging technologies
One of defining features of the cells of the nervous system is their strong polarity. They have long projections that extend to distances that could be thousands of times the length of the cell body. To support their function and to maintain homeostasis, the cells need to supply signals and resource...
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| Format: | Thesis |
| Language: | English |
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2023
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| _version_ | 1824458740824276992 |
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| author | Stoychev, D |
| author2 | Davis, I |
| author_facet | Davis, I Stoychev, D |
| author_sort | Stoychev, D |
| collection | OXFORD |
| description | One of defining features of the cells of the nervous system is their strong polarity. They have long projections that extend to distances that could be thousands of times the length of the cell body. To support their function and to maintain homeostasis, the cells need to supply signals and resources like proteins to such remote sites away from the nucleus. This is particularly important for dynamic processes such as cytoskeletal rearrangement, membrane trafficking, and synaptic plasticity. There is considerable evidence that at least some of this peripheral activity is regulated by the transport and local translation of mRNA molecules at the distant cytoplasm of neurons. The molecular mechanisms which govern the post-transcriptional regulation of these processes in neurons is still not fully understood, and even less is known in glia. This is partly due to the technically challenging aspect of capturing live mRNA dynamics and partly because only a few examples have been studied in detail. I begin to fill this technological gap by assessing and applying state-of-the-art methods from the fields of microscopy and image processing. This includes the use of adaptive optics for aberration correction and for remote (optical) focusing, 3D structured illumination microscopy for optical sectioning and for super-resolution, and the development of an open-source software platform that can enable all this functionality in fast complex experiments. On the image processing side, it includes the application of deep learning to the denoising of fluorescence image data for fast live imaging with minimised phototoxicity and photobleaching. Empowered by all these technological advancements I establish an improved live motility assay for the study of mRNA dynamics, using the <em>Drosophila</em> peripheral nervous system and especially the neuromuscular junction, which is arguably the simplest and easiest to use model to study these processes live. The MS2-MCP system is used to insert into mRNAs of interest special structures to which RNA-binding proteins, fused to a fluorescent protein such as GFP, can then bind. I apply this assay to investigate a specific mRNA, <em>shot</em>, previously identified as encoding important protein regulators of synaptic development and linked to neurodevelopmental disorders. |
| first_indexed | 2025-02-19T04:30:42Z |
| format | Thesis |
| id | oxford-uuid:db9c6447-6a7c-43ae-bbbf-d532246e1cd0 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-02-19T04:30:42Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:db9c6447-6a7c-43ae-bbbf-d532246e1cd02024-12-16T15:19:15ZElucidating the mechanism of mRNA transport using novel imaging technologiesThesishttp://purl.org/coar/resource_type/c_db06uuid:db9c6447-6a7c-43ae-bbbf-d532246e1cd0NeurosciencesBiologyDeep learning (machine learning)Optics, adaptiveMicroscopyEnglishHyrax Deposit2023Stoychev, DDavis, IBooth, MOne of defining features of the cells of the nervous system is their strong polarity. They have long projections that extend to distances that could be thousands of times the length of the cell body. To support their function and to maintain homeostasis, the cells need to supply signals and resources like proteins to such remote sites away from the nucleus. This is particularly important for dynamic processes such as cytoskeletal rearrangement, membrane trafficking, and synaptic plasticity. There is considerable evidence that at least some of this peripheral activity is regulated by the transport and local translation of mRNA molecules at the distant cytoplasm of neurons. The molecular mechanisms which govern the post-transcriptional regulation of these processes in neurons is still not fully understood, and even less is known in glia. This is partly due to the technically challenging aspect of capturing live mRNA dynamics and partly because only a few examples have been studied in detail. I begin to fill this technological gap by assessing and applying state-of-the-art methods from the fields of microscopy and image processing. This includes the use of adaptive optics for aberration correction and for remote (optical) focusing, 3D structured illumination microscopy for optical sectioning and for super-resolution, and the development of an open-source software platform that can enable all this functionality in fast complex experiments. On the image processing side, it includes the application of deep learning to the denoising of fluorescence image data for fast live imaging with minimised phototoxicity and photobleaching. Empowered by all these technological advancements I establish an improved live motility assay for the study of mRNA dynamics, using the <em>Drosophila</em> peripheral nervous system and especially the neuromuscular junction, which is arguably the simplest and easiest to use model to study these processes live. The MS2-MCP system is used to insert into mRNAs of interest special structures to which RNA-binding proteins, fused to a fluorescent protein such as GFP, can then bind. I apply this assay to investigate a specific mRNA, <em>shot</em>, previously identified as encoding important protein regulators of synaptic development and linked to neurodevelopmental disorders. |
| spellingShingle | Neurosciences Biology Deep learning (machine learning) Optics, adaptive Microscopy Stoychev, D Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title | Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title_full | Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title_fullStr | Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title_full_unstemmed | Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title_short | Elucidating the mechanism of mRNA transport using novel imaging technologies |
| title_sort | elucidating the mechanism of mrna transport using novel imaging technologies |
| topic | Neurosciences Biology Deep learning (machine learning) Optics, adaptive Microscopy |
| work_keys_str_mv | AT stoychevd elucidatingthemechanismofmrnatransportusingnovelimagingtechnologies |