A nucleotide binding-independent role for gamma-tubulin in microtubule capping and cell division

Berman et al. examine the capping activity of the gamma-TuRC. Using in vitro and cellular assays, they characterize how the gamma-TuRC's nucleotide-independent capping activity regulates microtubule dynamics and how it contributes to microtubule formation during cell division. The gamma-tubulin ring complex (gamma-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding gamma-TuRC-dependent microtubule nucleation, gamma-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human gamma-TuRC's capping activity. Single filament assays showed that the gamma-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of similar to 1 min. Reconstituted gamma-TuRCs with nucleotide-binding deficient gamma-tubulin (gamma-tubulin(Delta GTP)) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of gamma-tubulin suppressed non-centrosomal microtubule formation, add-back of gamma-tubulin(Delta GTP) could substantially restore this process. Our results suggest that gamma-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.