Analysis of microtubule-organizing centers (MTOCs) in Aspergillus nidulans and Neurospora crassa
Final Report Abstract
The eukaryotic microtubule cytoskeleton is required for chromosome segregation during nuclear and cell division and for other dynamic processes in the cell. Microtubule polymerization and its activity control is therefore crucial to most eukaryotic cells. Large protein complexes, known as microtubule-organizing centers (MTOCs) fulfill this function. In fungi one prominent MTOC is the spindle-pole body (SPB). In higher eukaryotes, and also in fungi, additional MTOCs were described. Whereas SPBs are embedded into the nuclear envelope one non-nuclear MTOC was discovered at septa of Aspergillus nidulans. The protein composition and the regulation of the activity of such septal MTOCs (sMTOCs) was largely unknown and was studied in this proposal. For the attachment of the protein complex we discovered a disordered protein, Spa10, which anchors sMTOCs at the septal pore. In human a new component of MTOCs was described some time ago and named MOZART. With the characterization of a MOZART orthologue, MztA, in A. nidulans we identified an unexpected complexity of the composition of the different MTOCs in hyphal compartments. Whereas the outer plaque of the SPB does not contain MztA and ApsB/Spa18 are used as receptors for gamma-tubulin small complexes, the inner plaque contains MztA and uses PcpA to anchor gamma-tubulin ring complexes. In sMTOCs MztA is present and ApsB/Spa18 is used to anchor gamma-tubulin ring complex. The biggest surprise was the discovery that the composition of the three different protein complexes was highly dynamic throughout the cell cycle. Analyses of the polymerization activity of SPBs and sMTOCs revealed coordination of the activity of the outer plaque of the SPBs and the sMTOCs. We identified the polo-like kinase PlkA and showed that it binds the gamma-tubulin ring complex receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the gamma-tubulin ring complex inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with gamma-tubulin ring complex receptors. The MT organization is very different in the related ascomycete N. crassa. Whereas in A. nidulans only 4-5 MTs are parallel organized and span through the compartment, in N. crassa many more MTs are present and form a network. All MTs are formed from the SPBs of the nuclei. The composition and organization of the SPBs appears to be comparable to the one in A. nidulans. However, at septa real MTOCs are missing, but MTs from neighbor compartments reach through the pore and form a pseudo-MTOC.
Publications
- (2017) Microtubule-organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein. Mol Microbiol 106: 285-303
Zhang Y, Gao X, Manck R, Schmid M, Osmani AH, Osmani SA, Takeshita N, Fischer R
(See online at https://doi.org/10.1111/mmi.13763) - (2019) Aspergillus nidulans als Modellsystem für filamentöse Pilze. In: Modellorganismen. Springer Spektrum, Berlin: pp: 93-116
Fischer R
(See online at https://doi.org/10.1007/978-3-662-54868-4_4) - (2019) The cytoskeleton and polarity markers during polarized growth of filamentous fungi. In: The Mycota. Springer, Heidelberg: pp: 43-62
Takeshita N and Fischer R
(See online at https://doi.org/10.1007/978-3-030-05448-9_3) - (2019) The spindle pole body of Aspergillus nidulans is asymmetrical and contains changing numbers of gamma-tubulin complexes. J Cell Sci 132: 234799
Gao X, Schmid M, Zhang Y, Fukuda S, Takeshita N, Fischer R
(See online at https://doi.org/10.1242/jcs.234799) - (2021) Application of PALM superresolution microscopy to the analysis of microtubule-organizing centers (MTOCs) in Aspergillus nidulans. Review. Methods Mol Biol 2329: 277-289
Gao X, Fischer R, Takeshita N
(See online at https://doi.org/10.1007/978-1-0716-1538-6_20) - (2021) The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control. J Cell Sci 134: jcs256537
Gao X, Herrero S, Wernet V, Erhardt S, Valerius O, Braus GH, Fischer R
(See online at https://doi.org/10.1242/jcs.256537) - (2022) The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa. Fungal Genet Biol 162: 103729
Ramirez-Cota, R, Espino-Vazquez AN, Rodriguez-Vega TC, Macias-Diaz RE, Callejas-Negrete OA, Freitag M, R. Fischer, Roberson RW and Mourino-Perez RR
(See online at https://doi.org/10.1016/j.fgb.2022.103729)
