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The HAUS6 CH domain binds microtubules to orient augmin for microtubule branching

ABSTRACT: Branching microtubule nucleation is a key mechanism for mitotic and meiotic spindle assembly and requires the hetero-octameric augmin complex. Augmin recruits the major microtubule nucleator, the γ-tubulin ring complex, to pre-existing microtubules to direct the formation of new microtubules in a defined orientation. Although recent structural work has provided key insights into augmin’s structural organization, molecular details of its interaction with microtubules remain elusive. Here, we identify the minimal conserved microtubule-binding unit of augmin across species and demonstrate that the low-complexity microtubule-binding unit from D. melanogaster mediates microtubule binding predominantly via the calponin homology (CH) domain in Dgt6/HAUS6. Comparative sequence and functional analyses reveal a highly conserved architecture of the HAUS6 CH domain and variations in the importance of the HAUS8 N-terminus in microtubule binding. Using cryo-electron microscopy and molecular dynamics simulations, we show that the HAUS6 CH domain binds at the inter-protofilament groove between two adjacent β-tubulin subunits and thereby orients augmin on microtubules. Altogether, our findings reveal how augmin binds microtubules to pre-determine the branching angle during microtubule nucleation and thereby facilitates the rapid assembly of complex microtubule networks.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Xenopus Laevis (african Clawed Frog) Arabidopsis Thaliana (mouse-ear Cress) Drosophila Melanogaster (fruit Fly)

SUBMITTER: Marcin Luzarowski

LAB HEAD: Marcin Luzarowski

PROVIDER: PXD060099 | Pride | 2025-07-30

REPOSITORIES: Pride

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			Action	DRS
	EC24-204-01.raw	Raw
	EC24-204-02.raw	Raw
	EC24-204-03.raw	Raw
	EC24-204-04.raw	Raw
	EC24-204-S01-S04_cs_ecoli_human.msf	Msf

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The augmin complex architecture reveals structural insights into microtubule branching

Project description:In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is strongly hampered by the lack of structural information on the augmin complex. Here we elucidate the molecular architecture and define the conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two Calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

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2021-11-01 | GSE186861 | GEO

CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

Project description:γ-tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here, we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus-ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends, but not the minus-end tracking protein CAMSAP1 induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits formation of CAMSAP2-bound microtubules detached from the microtubule-organizing center. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

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Structure of the native gamma-Tubulin Ring Complex capping spindle microtubules

Project description:Microtubules (MTs) are fundamental to cellular architecture, function and organismal development. They are nucleated from microtubule organizing centres by the evolutionary conserved ?-tubulin ring complex (?TuRC). However, the molecular mechanism of nucleation remains elusive. Here, we used cryo-electron tomography (cryo-ET) to determine the structure of the native ?TuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, ?TuRC presents a ring of ?-tubulin subunits to seed nucleation of exclusively 13-protofilament microtubules, and it adopts an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-ET reconstruction also revealed that a novel coiled-coil protein staples the first row of ?/?-tubulin molecules to alternating positions along the ?-tubulin ring. This positioning of ?/?-tubulin onto ?TuRC suggests a role for the coiled-coil protein in augmenting ?TuRC-mediated microtubule nucleation. Based on our results we describe a molecular model for budding yeast ?TuRC activation and MT nucleation.

2024-03-05 | PXD050372 | Pride

ZNF131 suppresses centrosome fragmentation in Glioblastoma stem-like cells through regulation of HAUS5

Project description:To identify new Glioblastoma multiforme (GBM) therapeutic strategies, we previously performed genome-wide RNAi lethality screens in patient-derived GBM stem-like cells (GSCs) and neural progenitor cells (NPCs) to identify genes required for the self-renewal of GSC isolates, but which are dispensable for NPCs. Here we report the retest of the GSC-lethal gene ZNF131, which encodes a novel vertebrate-specific BTB domain zinc finger transcription factor that is broadly required for GSC viability. Examination of gene expression changes after ZNF131 knockdown (kd) revealed that ZNF131 activity notably promotes expression of Joubert Syndrome ciliopathy genes, including KIF7, NPHP1, and TMEM237, as well as HAUS5, a component of Augmin/HAUS complex that facilitates microtubule (MT) nucleation along the mitotic spindle. Of these genes only kd of HAUS5 displayed GSC-specific viability loss, and, critically, HAUS5 ectopic expression was sufficient to suppress viability defects of ZNF131 kd cells. Moreover, ZNF131 and HAUS5 kd phenocopied each other in GSCs, each causing: mitotic arrest, centrosome fragmentation, loss of Augmin/HAUS complex on the mitotic spindle, and loss of GSC self-renewal and tumor formation capacity. In control NPCs, we observed centrosome fragmentation and lethality only when HAUS5 kd was combined with kd of HAUS2 or HAUS4, two other Augmin complex members, demonstrating that the complex is essential in NPCs, but that GSCs have heightened requirement. Our results suggest that GSCs differentially rely on ZNF131-dependent expression of HAUS5 as well as the Augmin/HAUS complex activity to maintain the integrity of centrosome function and viability. We speculate that this requirement likely arises from aneuploidy frequently observed in late stage GBM tumors, rather than supernumerary centrosomes.

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Sub-centrosomal mapping identifies augmin-TuRC as part of a centriole-stabilizing scaffold

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