The spindle pole body of Aspergillus nidulans is asymmetrical and contains changing numbers of γ-tubulin complexes

Abstract

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) are found in many cell types. Their composition and structure are only poorly understood. Here we analyzed nuclear MTOCs (spindle-pole bodies, SPBs) and septal MTOCs in Aspergillus nidulans. They both contain γ-tubulin along with a family of γ-tubulin complex proteins (GCPs). Our data suggest that SPBs consist of γ-tubulin small complexes (γ-TuSC) at the outer and larger γ-tubulin ring complexes (γ-TuRC) at the inner plaque. We show that an orthologue of the human MOZART protein interacted with the inner plaque receptor PcpAPcp1 at SPBs, while no interaction nor co-localization was detected between MztA and the outer plaque receptor ApsBMto1. Septal MTOCs consist of γ-TuRCs including MztA but are anchored through AspB and Spa18Mto2. MztA was not essential although abnormal spindles were observed frequently. Quantitative PALM imaging revealed unexpected dynamics of the protein composition of SPBs with changing numbers of γ-tubulin complexes over time during interphase and constant numbers during mitosis.

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

“…Furthermore, unlike direct stochastic optical resolution microscopy (dSTORM) in Lengefeld et al, 2018 , which presumed a model for MT formation in vivo based on in vitro work, our analysis made no assumptions and only took into account fluorescence intensity — a reliable readout of γTC abundance given the increase observed as a function of cell ploidy or during the cell cycle. In agreement with data presented here, studies in A. nidulans and S. pombe revealed heterogeneity in γTC levels at SPBs throughout interphase compared to more uniform levels in mitosis ( Bestul et al, 2017 ; Gao et al, 2019 ). Differences in the composition of γTC and its interactor, Mozart, as well as phosphoregulation of Mto2 (an activating subunit of the Spc72 ortholog, Mto1), are thought to underlie SPB asymmetry in these cases ( Borek et al, 2015 ; Gao et al, 2019 ).…”

“…In agreement with data presented here, studies in A. nidulans and S. pombe revealed heterogeneity in γTC levels at SPBs throughout interphase compared to more uniform levels in mitosis ( Bestul et al, 2017 ; Gao et al, 2019 ). Differences in the composition of γTC and its interactor, Mozart, as well as phosphoregulation of Mto2 (an activating subunit of the Spc72 ortholog, Mto1), are thought to underlie SPB asymmetry in these cases ( Borek et al, 2015 ; Gao et al, 2019 ). However, all of these factors are absent in budding yeast.…”

MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex

“…In an accompanying study, Huang et al (2020 [this issue of Cell Reports]) also report crystal structures for MZT modules and demonstrate that the MZT1 homolog in S. pombe is dispensable for targeting γ-TuRCs to spindle poles during mitosis but is still essential for cell viability. These observations have led to a model in which γ-TuRCs exist in multiple compositional states, even within the same cell type, allowing cells to tune the γ-TuRC from a microtubule nucleator to a microtubule anchor ( Gao et al, 2019 ; Muroyama et al, 2016 ; Sallee et al, 2018 ; Tovey et al, 2018 ). However, three recent, independent structures of vertebrate γ-TuRCs isolated using different affinity handles all display a well-defined lumenal bridge ( Consolati et al, 2020 ; Liu et al, 2020 ; Wieczorek et al, 2020 ).…”

“…MZT1 and MZT2 were subsequently identified in human γ-TuRCs ( Hutchins et al, 2010 ; Teixidó-Travesa et al, 2010 ), and both proteins have been consistently reported in biochemical analyses of vertebrate γ-TuRCs ( Choi et al, 2010 ; Consolati et al, 2020 ; Liu et al, 2020 ; Thawani et al, 2018 ; Wieczorek et al, 2020 ). Although MZT1 depletion in cultured cells phenocopies the effects of γ-Tubulin RNAi ( Hutchins et al, 2010 ), studies in fission yeast, C. elegans , D. melanogaster , A. nidulans , plant, and human cells have argued that MZTs are not required for γ-TuRC assembly; rather, they interact with GCPs to mediate γ-TuRC localization at specific microtubule organizing centers such as centrosomes ( Sallee et al, 2018 ; Teixidó-Travesa et al, 2010 ), inner plaques of spindle pole bodies ( Gao et al, 2019 ), basal bodies ( Tovey et al, 2018 ), or pre-existing microtubules ( Janski et al, 2012 ; Masuda et al, 2013 ; Nakamura et al, 2012 ). However, exactly how MZTs bind to γ-TuRCs is not clear.…”

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

“…In filamentous fungi, the minus ends of MTs locate at spindle pole bodies which serve as MT-organizing centers (MTOC). Additionally, areas close to the septum can act as septal MTOC (sMTOC) (Zekert et al 2010;Gao et al 2019). In the tip compartments, we observed dynamic MTs reaching the hyphal tip (Figure 8A).…”