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1. The γ-tubulin complex protein GCP6 is crucial for spindle morphogenesis but not essential for microtubule reorganization in Arabidopsis

   https://doi.org/10.1073/pnas.1912240116  

   Miao, Huiying; Guo, Rongfang; Chen, Junlin; Wang, Qiaomei; Lee, Yuh-Ru Julie; Liu, Bo (December 2019, Proceedings of the National Academy of Sciences)

   γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the GCP6 gene encoding the largest subunit of the γTuRC in Arabidopsis thaliana . Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. The gcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although the gcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. The gcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization. 

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2. Spatiotemporal organization of branched microtubule networks

   https://doi.org/10.7554/eLife.43890  

   Thawani, Akanksha; Stone, Howard A; Shaevitz, Joshua W; Petry, Sabine (May 2019, eLife)

   To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks. 

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3. Microtubules in Microorganisms: How Tubulin Isotypes Contribute to Diverse Cytoskeletal Functions

   https://doi.org/10.3389/fcell.2022.913809  

   Bera, Abesh; Gupta, Mohan L. (July 2022, Frontiers in Cell and Developmental Biology)

   The cellular functions of the microtubule (MT) cytoskeleton range from relatively simple to amazingly complex. Assembled from tubulin, a heterodimeric protein with α- and β-tubulin subunits, microtubules are long, hollow cylindrical filaments with inherent polarity. They are intrinsically dynamic polymers that utilize GTP binding by tubulin, and subsequent hydrolysis, to drive spontaneous assembly and disassembly. Early studies indicated that cellular MTs are composed of multiple variants, or isotypes, of α- and β-tubulins, and that these multi-isotype polymers are further diversified by a range of posttranslational modifications (PTMs) to tubulin. These findings support the multi-tubulin hypothesis whereby individual, or combinations of tubulin isotypes possess unique properties needed to support diverse MT structures and/or cellular processes. Beginning 40 years ago researchers have sought to address this hypothesis, and the role of tubulin isotypes, by exploiting experimentally accessible, genetically tractable and functionally conserved model systems. Among these systems, important insights have been gained from eukaryotic microbial models. In this review, we illustrate how using microorganisms yielded among the earliest evidence that tubulin isotypes harbor distinct properties, as well as recent insights as to how they facilitate specific cellular processes. Ongoing and future research in microorganisms will likely continue to reveal basic mechanisms for how tubulin isotypes facilitate MT functions, along with valuable perspectives on how they mediate the range of conserved and diverse processes observed across eukaryotic microbes. 

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4. Cryo-EM structures of plant Augmin reveal coiled-coil assembly, antiparallel dimerization, and NEDD1 binding

   https://doi.org/10.1038/s41467-025-66332-4  

   Ashaduzzaman, Md; Taheri, Aryan; Lee, Yuh-Ru Julie; Tang, Yuqi; Mittal, Shubham; Guo, Fei; Morcos, Faruck; Fried, Stephen D; Liu, Bo; Al-Bassam, Jawdat (December 2025, Nature Communications)

   Abstract Microtubule (MT) branch nucleation requires Augmin and NEDD1 proteins, which recruit and activate the gamma-tubulin ring complex (γ-TuRC). Augmin is a fork-shaped assembly of eight coiled-coil subunits, while NEDD1 is a β-propeller protein bridging MTs, Augmin, and γ-TuRC. We reconstituteArabidopsis thalianaAugmin assemblies and determine 3.7-7.3-Å cryo-EM structures of its V-junction and extended regions using crosslinking mass spectrometry. These structures reveal a complete plant Augmin model showing multi-coiled-coil interfaces stabilizing its 40-nm hetero-octameric fork architecture. The dual calponin homology (CH) domains at the V-junction terminus adopt open and closed conformations for MT binding. A 12-Å cryo-EM structure shows Augmin undergoes anti-parallel dimerization through conserved surfaces on its extended region. We determine the NEDD1 β-propeller structure with Augmin, revealing direct binding inside the V-junction that enhances dimerization. Direct coupling and evolutionary analyses identify co-varying residue pairs validating the eight-subunit model and NEDD1 interface. Cooperativity between dual CH domains and NEDD1 binding may regulate V-junction binding to MT lattices. This V-shaped dual binding anchors Augmin along MTs, creating platforms for γ-TuRC recruitment and branched MT nucleation. 

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5. In-Cell Association of a Bioorthogonal Tubulin

   https://doi.org/10.1021/acs.biomac.3c01253  

   Wang, Yuhan; Unnikrishnan, Mahima; Ramsey, Brooke; El Andlosy, Driss; Keeley, Alex T.; Murphy, Catherine J.; Gruebele, Martin (February 2024, Biomacromolecules)

   Studies of proteins from one organism in another organism’s cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro. 

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