Developing a LOVTRAP-based optogenetic reagent to disrupt intracellular dynein-dynactin interaction
| Autor: | Hsu, Li-Hui, 徐莉惠 |
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| Rok vydání: | 2019 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 107 Cytoplasmic dynein is a large protein complex that moves along the microtubule (MT) towards the minus-end via ATP hydrolysis. Dynein-related motility requires dynactin complex as a cofactor to link cellular cargoes to dynein motor, and this dynein-dynactin supercomplex is involved in a variety of important processes in eukaryotic cells. The interaction between dynein and dynactin complex is mediated by the interaction between the coil-coiled 1 region (CC1) of the p150Glued subunit in dynactin and the dynein intermediate chain (DIC) in dynein complex. We showed that overexpressing N-terminal of DIC (amino acid 1-237, DIC-N237) in cells can disrupt dynein-driven cellular organization, such as Golgi apparatus organization. This work aims to locally disrupt dynein-dynactin interaction without altering the overall dynein activity. To achieve this, an optogenetic tool termed PADDI (PhotoActivatable Dynein-Dynactin Interruptor) based on the LOVTRAP system was created. DIC-N237 is sequestered at outer membrane of mitochondria in the dark; upon light irradiation, it is released into the cytosol to disrupt dynein-dynactin interaction. Our preliminary results show that PADDI leads to global Golgi apparatus dispersion after cells were exposed to continuous and uniform light irradiation. However, we were unable to achieve local Golgi apparatus dispersion via pulsatile and focal light irradiation. The inability of PADDI to locally disperse Golgi apparatus may be related to the poor release of DIC-N237 from the mitochondria upon focal light irradiation. In addition, PADDI expression alters the distribution of the mitochondria indicating that sequestering N237-ZDK on the mitochondrial outer membrane may not be the ideal approach. Future endeavor will be focused on identifying a more suitable cellular target to sequester N237-ZDK on. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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