γ-TuRC Heterogeneity Revealed by Analysis of Mozart1

Abstract

SummaryMicrotubules are essential for various cell processes [1] and are nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs) at various microtubule organizing centers (MTOCs), including centrosomes [2, 3, 4, 5, 6]. Recruitment of γ-TuRCs to different MTOCs at different times influences microtubule array formation, but how this is regulated remains an open question. It also remains unclear whether all γ-TuRCs within the same organism have the same composition and how any potential heterogeneity might influence γ-TuRC recruitment. MOZART1 (Mzt1) was recently identified as a γ-TuRC component [7, 8] and is conserved in nearly all eukaryotes [6, 9]. Mzt1 has so far been studied in cultured human cells, yeast, and plants; its absence leads to failures in γ-TuRC recruitment and cell division, resulting in cell death [7, 9, 10, 11, 12, 13, 14, 15]. Mzt1 is small (∼8.5 kDa), binds directly to core γ-TuRC components [9, 10, 14, 15], and appears to mediate the interaction between γ-TuRCs and proteins that tether γ-TuRCs to MTOCs [9, 15]. Here, we use Drosophila to investigate the function of Mzt1 in a multicellular animal for the first time. Surprisingly, we find that Drosophila Mzt1 is expressed only in the testes and is present in γ-TuRCs recruited to basal bodies, but not to mitochondria, in developing sperm cells. mzt1 mutants are viable but have defects in basal body positioning and γ-TuRC recruitment to centriole adjuncts; sperm formation is affected and mutants display a rapid age-dependent decline in sperm motility and male fertility. Our results reveal that tissue-specific and MTOC-specific γ-TuRC heterogeneity exist in Drosophila and highlight the complexity of γ-TuRC recruitment in a multicellular animal.

Subcellular localization and mitotic interactome analyses identify SIRT4 as a centrosomally localized and microtubule associated protein

“…S12) with -tubulin, TUBGCP2, and TUBGCP3, which represent core components of the TURC. The latter is located at the outer region of the pericentriolar material (PCM) 66,67 and functions as nucleator of microtubules at their minus poles 49,50 . It remains to be determined whether SIRT4 directly or indirectly regulates recruitment of the TURC to centrosomes or its microtubule nucleation activity.…”

“…4, we identified several mitochondrial SIRT4-binding proteins and potential substrates (OPA1 42 ; ATP5F1A 34 ; ANT2 21 ; IDE 48 ) as well as mostly novel, extra-mitochondrial localized SIRT4 interactors. The latter comprise -and -tubulin as subunits of microtubules, components of the centrosomally localized TURC complex (-tubulin, TUBGCP2, TUBGCP3) 49,50 that nucleates microtubules at their minus poles, the microtubule deacetylase HDAC6 that is critically involved in the regulation of microtubule stability and dynamics 9 , and the G2/M cell cycle regulator CDK1 51 . These SIRT4 interactions were confirmed by nanobody-mediated coimmunoprecipitation analyses (Fig.…”

Microtubules originate asymmetrically at the somatic Golgi and are guided via Kinesin2 to maintain polarity within neurons

“…3B,C; Fig. S2), which are typically scattered throughout the cytosol in Drosophila cells while still maintaining cismedial-trans polarity 58 . These Golgi stacks can be oriented either side-on or face-on to the imaging plane, appearing either more elongated or circular, respectively.…”

“…Golgi outposts are enriched at dendritic branch points 54,55 and studies in Drosophila suggest that microtubules nucleated from Golgi outposts help regulate dendritic branching and contribute to maintaining microtubule polarity 12,41,42 . Unlike the somatic Golgi, which in both mammalian cells and Drosophila comprises cis, medial and trans compartments organised into stacks and ribbons 56,58 , Golgi outposts can be either single-or multicompartment units with multi-compartment units having a higher propensity to initiate microtubule growth 42 . Within class I da neurons, Golgi outpost-mediated nucleation is thought to be dependent on the γ-TuRC-tethering protein Cnn and is thought to help restrict dendritic branching 41 , while within class IV neurons Golgi outpost-mediated nucleation is thought to be dependent on Pericentrin-like protein (Plp) and is thought to help promote dendritic branching 12 .…”

“…The multi-serine insert acts as a flexible linker between γ-tubulin23C and eGFP. The following guide RNA sequences were used to cut sfGFP-Cnn-P1 (insertion of sfGFP at the start of exon 1a) and sfGFP-Cnn-P2 (insertion of sfGFP at the start of exon 1b) alleles were generated within the lab following the approach used previously58 . Briefly, flies expressing a single guide -P2) were crossed to nos-Cas9 expressing females and the resulting embryos were injected with a pBluescript plasmid containing sfGFP and linker sequence (4X GlyGlySer) flanked on either side by 1.5kb of DNA homologous to the cnn genomic locus surrounding the 5' end of the appropriate coding region.…”