Seminar Title: Structural and biochemical studies reveal principles of microtubule nucleation
Tarun Kapoor, Ph.D.
Pels Family Professor
The Rockefeller University
Seminar abstract: Assembly of the cell division apparatus depends on the 𝛾-tubulin ring complex (𝛾-TuRC), an essential regulator of centrosomal and non-centrosomal microtubule formation. Our structural studies of this ~2.3 MDa complex reveal how >31 proteins, including 𝛾-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB) are organized into an asymmetric cone-shape architecture. We have also biochemically reconstituted the human 𝛾-TuRC (𝛾-TuRC-GFP), a ~35S complex that nucleates microtubules in vitro. In addition, we have characterized a subcomplex, 𝛾-TuRCΔLB-GFP, which lacks MZT1 and actin. Remarkably, we find that 𝛾-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar kinetics as the holocomplex. Electron microscopy reveals that 𝛾-TuRC-GFP resembles the native 𝛾-TuRC architecture, while 𝛾-TuRCΔLB-GFP adopts a partial cone shape presenting only 8-10 𝛾-tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” within the 𝛾-TuRC. Together, recombinant forms of human 𝛾-TuRC, a/b-tubulin and the hetero-octameric augmin complex will help define the critical components and uncover the basic principles of microtubule formation during cell division.