- Sanders AAWM, Chang K, Zhu X, Thoppil RJ, Holmes WR, Kaverina I. Non-random γ-TuNA-dependent spatial pattern of microtubule nucleation at the Golgi. Molecular biology of the cell. 2017 Sep 20. PMID: 28931596 [PubMed].
Non-centrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from non-random nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "Cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "Heterogeneous Golgi" model, MT nucleation is dramatically upregulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally described by both models, simulating MT length distributions within the "Cooperativity" model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizers CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites.