The University of Manchester Doctor of Philosophy Using the BioID Method to Investigate the Dynein Light Intermediate Chain Interactome Amy Davidson 22/09/2017 Cytoplasmic dynein 1 is a large multi-subunit complex that has a multitude of functions and therefore, many interactions. Dynein has two light intermediate chains (LICs) that bind in a mutually exclusive manner to the heavy chain and are proposed to form two distinct populations of dynein that could bind to different cargoes. Both unique and shared interactions have been discovered for the two LICs. Identifying the interactions of the LICs is hindered by the large size of the dynein complex, and if immunoprecipitation or pull-down approaches are used, by the need to retain the interactor-complex link through subsequent cell lysis and purification. We used the novel BioID method in an attempt to identify potential new LIC interactors, be they unique or shared. The BioID method utilises a mutated biotin ligase, BirA*, that generates activated biotin that then labels proteins in its vicinity. These biotinylated proteins can then be purified using streptavidin. The LICs were tagged with Myc BirA* on both termini and we confirmed that this tag had no dominant negative effect on dynein functions, such as positioning the Golgi apparatus. The tagged LICs were able to rescue the effects caused by a double endogenous LIC knockdown, therefore suggesting that they are functional and part of the dynein complex. Stable cell lines of Myc BirA* LIC1, Myc BirA* LIC2, LIC2 Myc BirA* and Myc BirA* GFP were generated. The tagged LICs in the stable cell lines could still rescue a double endogenous LIC knockdown whereas the Myc BirA* GFP did not. We tested the amount of incorporation of the tagged LICs into the dynein complex by sucrose density gradient centrifugation to try and limit unspecific biotin labelling. Our BioID mass spectrometry data for the tagged LICs revealed several dynein and dynactin subunits, demonstrating successful incorporation of the LICs into intact dynein complexes that could interact with dynactin. In addition, we identified the known dynein-dynactin adaptor protein, BicD2. Several other proteins of potential interest were identified, such as Importin 7 and Mre11. Further experiments are needed to confirm these as LIC near-neighbours and to then validate them as direct interactors.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Viki Allan (Supervisor) & Jean-Marc Schwartz (Supervisor)|
Using the BioID Method to Investigate the Dynein Light Intermediate Chain Interactome
Davidson, A. (Author). 1 Aug 2018
Student thesis: Phd