Breaking Down Microtubule Formation: Characterizing the Biochemical and Cellular Functions of the γ-Tubulin Ring Complex

Author

Adi Y. Berman

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Kapoor Laboratory

Abstract

The γ-Tubulin ring complex (γ-TuRC) is an essential regulator of the microtubule cytoskeleton. It is composed of>30 individual proteins that include the major component, γ-tubulin, as well as γ-tubulin complex proteins 2-6 (GCP2-6), mitotic-spindle organizing proteins associated with a ring of γ-tubulin proteins 1 and 2 (MOZART, or MZT1 and MZT2), and an actin molecule. This ~2.2MDa assembly regulates microtubule dynamics by facilitating the nucleation of new microtubules, modulating microtubule minus-end dynamics by acting as a minus-end cap, and anchoring microtubules to specify their cellular localization. These three major activities of the γ-TuRC, nucleation, capping, and anchoring, contribute to the dynamic nature of individual microtubules and of larger microtubule networks that are critical for cellular activities, including cell motility, in trace have spurred biochemical and cellular characterization of its nucleation activity. However, it remains unclear how the remaining major γ-TuRC activities contribute to microtubule dynamics. In the first part of this thesis, I characterize the capping activity of the γ-TuRC. Using biochemical assays, I examine the association of recombinantly expressed and purified γ-TuRC at microtubule minus-ends either following a nucleation event, or on pre-formed microtubules. Using total internal reflection fluorescence (TIRF) microscopy, the dynamics of this association can be quantified in terms of the number of microtubules that are capped, and the length of time the cap persists at the minus-end under these two conditions. Additionally, I purified a recombinant γ-TuRC composed of GTP-binding deficient γ-tubulin in order to examine the GTP-binding dependency of the γ-TuRC’s capping activity. As opposed to the γ-TuRC’s nucleation activity, which is GTP-binding dependent, I found that the γ-TuRC’s capping activity is GTP-binding independent. By expressing GTP-binding deficient γ-tubulin in HeLa cells depleted of endogenous γ-tubulin protein, I characterized the role of the γ-TuRC’s capping activity in dividing cells. While cells expressing the GTP-binding γ-tubulin mutant could not form bipolar mitotic spindles and became arrested in mitosis, fixed- and live-cell imaging experiments showed that expression of this mutant rescued non-centrosomal microtubule formation, which was lost under γ-tubulin knockdown conditions. Together, these data suggest that the γ-TuRC’s capping activity is GTP-binding independent and plays a role in non-centrosomal microtubule formation during mitosis. In the second part of my thesis, I perform studies towards further characterizing the γ-TuRC. First, I use affinity-purification followed by mass spectrometry to characterize the composition of γ-TuRCs purified from a HeLa cell line overexpressing GFP-tagged γ-tubulin. Further mass spectrometry analysis identified interacting proteins that co-purified with γ-tubulin, some of which have a known function related to the microtubule cytoskeleton or cell division. Second, I performed cell biology experiments to examine how the composition of the γ-TuRC may affect its activities.Recent work has suggested that the N-terminal domains of GCP6, named the N-helical domains (NHD) and the “belt”domain, are needed to maintain the structural integrity of the γ-TuRC, and that without these domains, specific components of the complex are lost. Cells expressing these N-terminally truncated GCP6 constructs displayed partial γ-tubulin-containing complexes relative to cells expressing full-length GCP6. Furthermore, cells expressing GCP6 truncated of the NHDs and the belt domain showed loss of centrosomal GCP6 and γ-tubulin specifically during mitosis, while their centrosomal localization persisted during interphase. Together, these data suggest that different components of the γ-TuRC may mediate the localization and anchoring of the γ-TuRC at specific cellular sites, such as the centrosome.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Berman, Adi Y., "Breaking Down Microtubule Formation: Characterizing the Biochemical and Cellular Functions of the γ-Tubulin Ring Complex" (2024). Student Theses and Dissertations. 752.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/752