Investigating the regulators of cytoplasmic dynein
| dc.contributor.author | Dilsaver, Matthew, author | |
| dc.contributor.author | Markus, Steven, advisor | |
| dc.contributor.author | Di Pietro, Santiago, committee member | |
| dc.contributor.author | DeLuca, Jennifer, committee member | |
| dc.contributor.author | Argueso, Juan Lucas, committee member | |
| dc.date.accessioned | 2020-01-13T16:42:16Z | |
| dc.date.available | 2022-01-07T16:41:53Z | |
| dc.date.issued | 2019 | |
| dc.description.abstract | Organization of the cell is a dynamic and complex process that is often underappreciated. To accomplish this, cells use motor proteins to move different cargo to their destination. Cytoplasmic dynein is one such motor protein that uses filaments called microtubules as tracks. However, there is only one cytoplasmic dynein to accomplish over forty tasks. To achieve this, the cell uses a complex array of cofactors and regulators to specifically control dynein. But the role of each of these cofactors and regulators in poorly understood. To better understand how dynein is regulated we turn to budding yeast that provides a simplified system where dynein only has one known function, this is to position the spindle in the division plane between two dividing cells. Localizing dynein is extremely important. One regulator of dynein is Pac1 which was recently found to also activate dynein motility in vitro. Pac1 works to localize dynein to microtubule plus ends where it can interact with dynactin and Num1. Ndl1 is known to interact with Pac1, knockouts of Ndl1 led to a mild phenotype mimicking a dynein knockout. But how Ndl1 functions is poorly understood. Dynactin is an essential regulator of almost all dynein's tasks in humans and dynein's only role in yeast. Without dynactin, dynein cannot reach Num1 patches at the cell periphery and pull the spindle. In this study we sought to better understand dynactin and Ndl1's role in dynein regulation using in vitro single molecule assays where the activity of dynein can be recorded. Initial attempts to purify dynactin for these assays failed. We then developed a cell lysis assay to study dynactin and other proteins role in dynein regulation. We found in preliminary results that dynactin increased dynein activity. We also attempted to use a protein known as Num1, that is essential to dynein localization and interacts with dynactin, to purify the dynein-dynactin complex. Preliminary results showed that this complex was motile, indicating an intact complex. We also found that Ndl1 can bind motile dynein and increase run length using in vitro assays. We also were able to determine that Pac1 cannot bind dynein and Ndl1 at the same time indicating that there is a release mechanism for Pac1 from Ndl1 to bind dynein. We were able to map Ndl1's binding site to the N terminus of the dynein accessory chain Pac11. Then we tested to see if Ndl1 influence on Pac1-dynein interaction and found that Ndl1 was able to increase Pac1 comigrating with dynein in these assays. This work has opened new strategies for studying the regulators of dynein as well as better determined the interaction between Ndl1, dynein and Pac1. Further work will determine how each of these proteins affect dynein activity. | |
| dc.format.medium | born digital | |
| dc.format.medium | masters theses | |
| dc.identifier | Dilsaver_colostate_0053N_15842.pdf | |
| dc.identifier.uri | https://hdl.handle.net/10217/199862 | |
| dc.language | English | |
| dc.language.iso | eng | |
| dc.publisher | Colorado State University. Libraries | |
| dc.relation.ispartof | 2000-2019 | |
| dc.rights | Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright. | |
| dc.title | Investigating the regulators of cytoplasmic dynein | |
| dc.type | Text | |
| dcterms.embargo.expires | 2022-01-07 | |
| dcterms.embargo.terms | 2022-01-07 | |
| dcterms.rights.dpla | This Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). | |
| thesis.degree.discipline | Cell and Molecular Biology | |
| thesis.degree.grantor | Colorado State University | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.S.) |
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