Biology subjects

Munoz-Hernandez, H.

Publications and source records attributed to Munoz-Hernandez, H..

5 recordsLinked to original sources

bioRxiv2026

An architectural switch in the evolution of the γ-tubulin ring complex

Canonical microtubules contain 13-protofilaments and are templated by the {gamma}-tubulin ring complex ({gamma}-TuRC). However, some eukaryotes assemble non-canonical microtubules, like the 11-protofilament structures found in Caenorhabditis elegans. How {gamma}-TuRCs adapt to template alternative microtubule geometries is unclear. Here, we present the cryo-electron microscopy structure of the C. elegans {gamma}-TuRC ({gamma}-TuRCCe), revealing a cone-shaped assembly consistent with an 11-protofilament template. While the complex incorporates the conserved subunits actin, GCP2 and GCP3, {gamma}-TuRCCe replaces GCP4-6 with a divergent 4-spoked assembly containing additional copies of GCP2 and the nematode-specific proteins GTAP-1 and GTAP-2. Structures of nucleotide-free {gamma}-TuRCCe subcomplexes reveal partial {gamma}-tubulin unfolding, suggesting nucleotide binding stabilizes eukaryotic tubulins. Remarkably, reconstituted 4-spoked assemblies can multimerize into [~]13-fold symmetric microtubule nucleation templates in vitro, contrasting with the native complexs 11-protofilament architecture. Our work defines the structural blueprint of an 11-protofilament microtubule template and shows how divergent {gamma}-tubulin components are repurposed to accommodate non-canonical microtubule lattices.

biochemistry↗

bioRxiv2026

Structural basis of microtubule destabilization by GTP hydrolysis

Microtubules are cytoskeletal filaments that dynamically grow and shrink to support vital biological functions. Their behavior is regulated by GTP hydrolysis in tubulin incorporated into the lattice, but the underlying chemical transitions and their impact on microtubule stability remain unclear. Using cryo-electron microscopy, we resolved microtubule structures at 1.9-2.2 [A] in four different nucleotide states, revealing the roles of amino acids, ions, and hundreds of water molecules in the hydrolysis process. We found that compaction of the GTP-bound lattice into a more stable pre-hydrolysis state activates a catalytic water molecule for {gamma}-phosphate cleavage. Hydrolysis intermediates are stabilized by strengthened lattice contacts, but in the final GDP state, release of reaction products disrupts these contacts and destabilizes the lattice. Our results explain how chemical changes in tubulins nucleotide site drive lattice transitions and offer an atomistic framework for understanding microtubule dynamic instability.

biochemistry↗

bioRxiv2026

A cryo-EM processing pipeline for microtubules using CryoSPARC

Microtubules are cytoskeletal filaments typically characterized by a discontinuous helical lattice of /{beta}-tubulin heterodimers. Microtubules can also adopt variable lattice architectures both in vitro and in cellular contexts. Pseudo-helical averaging processing strategies have been developed to generate cryo-EM reconstructions of microtubules with and without decorating protein-binding partners, but these pipelines can be difficult to implement for the average user, especially for undecorated filaments. Here, we describe MiCSPARC, a cryo-EM processing pipeline developed around CryoSPARC (Punjani et al., 2017), which leverages automated particle picking and fast 3D refinement times in CryoSPARC to determine structures of both decorated and undecorated microtubules. We generated reconstructions of undecorated GDP microtubules, as well as kinesin-1 motor domain-decorated GMPCPP filaments at resolutions of up to 2.8 [A], demonstrating the robustness of the pipeline. Based on its convenient implementation and ability to routinely generate high-resolution, seam-corrected microtubule reconstructions, MiCSPARC should provide a valuable tool for understanding microtubule dynamics, microtubule-associated proteins, and microtubule-targeting agents.

biophysics↗

bioRxiv2023

Closure of the gamma-tubulin ring complex by CDK5RAP2 activates microtubule nucleation

Microtubule nucleation in cells is templated by the {gamma}-tubulin ring complex ({gamma}-TuRC), a 2.3 MDa multiprotein assembly concentrated at microtubule organizing centers (MTOCs). Current {gamma}-TuRC structures exhibit an open conformation that deviates from the geometry of /{beta}-tubulin in the microtubule, potentially explaining their low in vitro microtubule-nucleating activity. Several proteins have been proposed to activate the {gamma}-TuRC, but the mechanisms underlying activation are not known. Here, we isolated the porcine {gamma}-TuRC using CDK5RAP2s centrosomin motif 1 (CM1) and determined its structure with cryo-electron microscopy. 3D heterogeneity analysis revealed an unexpected conformation of the {gamma}-TuRC, in which five protein modules containing MZT2, GCP2, and CDK5RAP2 decorate the outer face of the holocomplex. These decorations drive a long-range constriction of the {gamma}-tubulin ring, bringing the GCP2/GCP3-rich core of the complex in close agreement with the architecture of a microtubule. A purified CDK5RAP2 fragment stimulated the microtubule nucleating-activity of the porcine {gamma}-TuRC as well as a reconstituted, CM1-free human complex in single molecule assays. Our results show that CDK5RAP2 activates the {gamma}-TuRC by promoting {gamma}-tubulin ring closure, providing a structural mechanism for the regulation of microtubule nucleation by CM1 motif proteins in mammals and revealing conformational transitions in {gamma}-tubulin that prime it for templating microtubule nucleation at MTOCs.

biochemistry↗

bioRxiv2020

Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone

The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with HSP90, functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, snRNPs and PI3 kinase-like kinases (PIKK) such as TOR and SMG1. PIKK stabilisation depends on an additional complex of TELO2, TTI1 and TTI2 (TTT), whose structure and function are poorly understood. We have now determined the cryo-EM structure of the human R2TP-TTT complex that together with biochemical experiments reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.

biochemistry↗