About the Project
The microtubule motors kinesin-1 and cytoplasmic dynein move many cargoes within cells, and this transport is crucial for life and health. For kinesin-1, the motor subunits and light chains (KLCs) can each interact with cargo, and the presence of three heavy chains (KIF5A, B and C) and four KLC genes provides a potential mechanism for directing the motor to different cargoes. While this model is appealing, and is supported by a number of studies, it has never been tested systematically. Moreover, little is known as yet about the role of KLCs 3 and 4, even though all four KLCs are expressed in most tissues and cell lines.
We will use a systematic mass spectrometry-based approach using proximity biotinylation (BioID) to identify interactors and cargoes of kinesin-1 isoform. In this approach, the each KIF5 and KLC isoform is fused with a biotin ligase, BirA, which generates activated biotin that will covalently attach to nearby proteins. These near-neighbours are isolated using streptavidin beads and then are identified by mass spectrometry. We have already made stable cell lines expressing BirA-tagged KLC1, KLC2 and KLC3, and will start by using total cell lysates to identify interactors. Further refinements would be compare membrane and cytosolic fractions, or mitotic vs. interphase extracts. We will select key interactors for futher studies. We will also use BirA-tagged KIF5A-C to investigate their specific interators.
Candidates are expected to have, or about to obtain, an upper second class (or equivalent) undergraduate degree in a related subject area. A Masters qualification in a similar area would be an advantage.
https://www.research.manchester.ac.uk/portal/en/persons/victoria-allan%28e61e2dd2-b6f1-4765-8532-4fff8843144d%29.html
Funding Notes
This project has a Band 2 fee. Details of our different fee bands can be found on our website (https://www.bmh.manchester.ac.uk/study/research/fees/). For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (https://www.bmh.manchester.ac.uk/study/research/apply/). Informal enquiries may be made directly to the primary supervisor.
References
1) Roux K, Kim D, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196: 801-810
2) Ruane, P. T., Gumy, L. F., Bola, B., Anderson, B., Wozniak, M. J., Hoogenraad, C. C., & Allan, V. J. (2016). Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2. Scientific Reports, 6, 27456.
3) Wozniak, MJ, Bola, B, Brownhill, K, Yang, Y-C, Levakova, V and Allan, VJ. (2009) Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells. J. Cell Sci. 122: 1979-1989