Microtubules are born and reborn continuously, even during quiescence. These polymers are nucleated from templates, namely γ-tubulin ring complexes (γ-TuRCs) and severed microtubule ends. Using single-molecule biophysics, we show that nucleation from γ-TuRCs, axonemes and seed microtubules requires tubulin concentrations that lie well above the critical concentration. We measured considerable time lags between the arrival of tubulin and the onset of steady-state elongation. Microtubule-associated proteins (MAPs) alter these time lags. Catastrophe factors (MCAK and EB1) inhibited nucleation, whereas a polymerase (XMAP215) and an anti-catastrophe factor (TPX2) promoted nucleation. We observed similar phenomena in cells. We conclude that GTP hydrolysis inhibits microtubule nucleation by destabilizing the nascent plus ends required for persistent elongation. Our results explain how MAPs establish the spatial and temporal profile of microtubule nucleation.

Highlightsd High resolution cryo-EM structure of the native human gtubulin ring complex (g-TuRC)d Identification and structural models for g-tubulin, GCP2, GCP3, GCP4, GCP5, and GCP6d Identification of an actin-like protein in the g-TuRC lumen d Insights into the regulation of microtubule nucleation by the g-TuRC

The structure and function of the synthetic innate defense regulator peptide 1018 was investigated. This 12 residue synthetic peptide derived by substantial modification of the bovine cathelicidin bactenecin has enhanced innate immune regulatory and moderate direct antibacterial activities. The solution state NMR structure of 1018 in zwitterionic dodecyl phosphocholine (DPC) micelles indicated an α-helical conformation, while secondary structures, based on circular dichroism measurements, in anionic sodium dodecyl sulfate (SDS) and phospholipid vesicles (POPC/PG in a 1:1 molar ratio) and simulations revealed that 1018 can adopt a variety of folds, tailored to its different functions. The structural data are discussed in light of the ability of 1018 to potently induce chemokine responses, suppress the LPS-induced TNF-α response, and directly kill both Gram-positive and Gram-negative bacteria.

SUMMARY Microtubule organization depends on the γ-Tubulin ring complex (γ-TuRC), a ~2.3-MDa nucleation factor comprising an asymmetric assembly of γ-Tubulin and GCP2-GCP6. However, it is currently unclear how the γ-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human γ-TuRC. MZT1 forms two subcomplexes with the N-terminal α-helical domains of GCP3 or GCP6 (GCP-NHDs) within the γ-TuRC “lumenal bridge.” We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native γ-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the γ-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic “modules” that can expand structural and regulatory interfaces in the γ-TuRC.

SUMMARYThe γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation 1–4. Metazoan γ-TuRCs isolate as ∼2 MDa complexes containing the conserved proteins γ-tubulin, GCP2 and GCP3, as well as the expanded subunits GCP4, GCP5, and GCP6 3,5,6. However, in current structural models, γ-TuRCs assemble solely from subcomplexes of γ-tubulin, GCP2 and GCP3 7. The role of the metazoan-specific subunits in γ-TuRC assembly and architecture are not currently known, due to a lack of high resolution structural data for the native complex. Here, we present a cryo-EM structure of the native human γ-TuRC at 3.8Å resolution. Our reconstruction reveals an asymmetric, single helical-turn and cone-shaped structure built from at least 34 polypeptides. Pseudo-atomic models indicate that GCP4, GCP5 and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes and are distal to the γ-TuRC “seam”. Evolutionary expansion in metazoan-specific subunits diversifies the γ-TuRC by introducing large (>100,000 Å2) surfaces that could interact with different regulatory factors. We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric composition and architecture, the human γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g. within biological condensates 8 or alongside existing filaments 9.