Characterisation of microtubule lumenal filaments using cryo-electron tomography

Abstract

Research on the structure and function of microtubules has classically focused on their assembly, dynamics, and interaction of the exterior wall with microtubule associated proteins. In comparison, the lumen of the microtubule has been relatively unexplored. Once considered hollow, an increasing number of electron microscopy studies have shown that the microtubule lumen can be occupied by a range of globular densities which vary in abundance and morphology depending on cell type and cell localisation; some early studies suggested the possibility that a subset of lumenal densities may in fact be a central filament, whereas others observed columns of particles and more isolated structures. Recently, actin filaments were observed inside the microtubule lumen in a small-molecule modified cell protrusion system using cryo-electron microscopy. This suggested the possibility that at least some of the previously observed lumenal material could be filamentous or globular actin. This also
suggested a potential novel mode of interaction between the two cytoskeletal proteins.
Throughout my thesis, I further characterise the lumenal filaments and indicate that they are highly heterogeneous in their morphology and protein composition. I show that the majority of filaments are composed of F-actin bound to its regulator cofilin. However, some lumenal filaments are revealed for the first time to be filaments assembled from metabolic enzymes. I reconstitute lumenal F-actin and cofilin-bound F-actin in vitro, laying the groundwork for future mechanistic studies to determine the effects of lumenal filaments on microtubule properties. Finally, I show for that microtubule lumenal F-actin and cofilin-bound F-actin exist in human platelets, showing that these lumenal filaments can be found in physiological settings without any cellular perturbations. Overall, my work shows the importance of considering the microtubule lumenal space as an environment where many different proteins could reside and function, and where they may have important effects on microtubule dynamics and function.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Mark P Dodding (Supervisor) & Paul Verkade (Supervisor)