Microbiology

First cell-end marker proteins in filamentous fungi

The establishement and maintenance of polarity is a common theme in Biology. Rod-shaped bacteria, the pollen tube in plants or neurons are text-book examples. Filamentous fungi are excellent models to study the molecular biology underlying polarity.
Fischer
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Cell end marker complexes are dynamic and define the area of growth

During the analysis of the KipA kinesin in Aspergillus nidulans we noticed that hyphae of a ∆kipA-mutant strain do not grow as straight as hyphae of wild type. Hence, KipA is required for polarity maintenance. This was the starting point for very successful research on cell-end marker proteins, some of which are transported by KipA. The field was greatly advanced by Norio Takeshita, a postdoc in the lab and meanwhile professor in Tsukube, Japan. The application of super resolution microscopy revealed a very dynamic picture of the cell-end marker complexes and a novel view of fungal hyphal extension.

Konzack, S., Rischitor, E. P., Enke, C. & Fischer, R. (2005) The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. Mol. Biol. Cell, 16:497-506.

Takeshita, N., Higashitsuji, Y., Konzack, S. & Fischer, R. (2008) Apical sterol-rich membranes are essential for localizing cell end markers that determine growth directionality in the filamentous fungus Aspergillus nidulans. Mol. Biol. Cell, 19(1):339-351.

Higashitsuji, Y., Herrero, S., Takeshita, N. & Fischer, R. (2009) The cell end marker protein TeaC is involved in growth directionality and septation in Aspergillus nidulans. Eukaryot. Cell, 8:957-967.

Takeshita, N., Mania, D., Herrero de Vega, S., Ishitsuka, Y., Nienhaus, G.U., Podolski, M., Howard, J. & Fischer, R. (2013) The cell end marker TeaA and the microtubule polymerase AlpA contribute to mirotubule guidance at the hyphal tip cortex of Aspergillus nidulans for polarity maintenance. J Cell Sci, 126:5400-5411.

 Ishitsuka, Y., Savage, N., Li, Y., Bergs, A., Kohler, D., Donnelly, R., Nienhaus, U., Fischer, R. & Takeshita, N. (2015) Super-resolution microscopy reveals a dynamic picture of cell polarity maintenance during directional growth. Science Advances, 1:e1500947.

Takeshita, N., Evangelinos, M., Zhou, L., Somera-Fajardo, R.A., Lu, L., Takaya, N., Nienhaus, G.U. & Fischer, R. (2017) Pulses of Ca2+ coordinate actin assembly and exocytosis for stepwise cell extension. Proc. Natl. Acad. Sci., 114:5701-5706

Phytochrome in fungi

Every biologists remembers phytochrome and its red-far red light absorption properties from the Botany classes. The discovery of pyhtochrome in bacteria and soon after in fungi was against the dogma that phytochrome is a plant molecule.
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Phytochrome-dependent red light sensing requires the HOG pathway.

It was well known that Aspergillus nidulans responds to red light. The discovery of phytochrome started with a bet in the lab when we were searching for a new subject for a Diploma thesis. At that time, in 2004, we were invovled in the annotation of the genome and Kay Vienken found a sequence in the genome with similarity to plant phytochrome. He suggested that we follow that discovery. I suggested a more "safe" project on a kinase protein and we bet which project would be more successful. Well, the phytochrome indeed turned out to be THE red-light sensor in A. nidulans and we are working very successfully on that since then.

After the characterization of the gene in A. nidulans and the expression of the phytochrome protein in E. coli, we analyzed the signal transduction cascade and discovered that phytochrome has a cytoplasmic and a nuclear function. In the nucleus it interacts with the blue-light receptor LreA and the transcription factor VeA. The cytoplasmic function was discovered in a forward genetics approach, where we isolated "blind" mutants. Whole genome sequencing of one of the mutant revealed a mutation in the MAP kinase HogA (SakA). This was a great discovery, because it showed that light and stress sensing is related. This explained also the role for light sensing in nature.

Blumenstein, A., Vienken, K., Tasler, R., Purschwitz, J., Veith, D., Frankenberg-Dinkel, N. & Fischer, R. (2005) The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr. Biol., 15:1833-1838.

Purschwitz, J., Müller, S., Kastner, C., Schöser, M., Haas, H., Espeso, E.A., Atoui, A., Calvo, A.M. & Fischer, R. (2008) Functional and physical interaction of blue and red-light sensors in Aspergillus nidulans. Curr. Biol., 18:255-259.

Hedtke, M., Rauscher, S., Röhrig ,J., Rodriguez, J., Yu, Z., & Fischer, R. (2015) Light-dependent gene activation in Aspergillus nidulans is strictly dependent on phytochrome and involves the interplay of phytochrome and white-collar-regulated histone H3 acetylation. Mol. Microbiol., 97:733-745.

Rauscher, S., Pacher, S., Kniemeyer, O. & Fischer, R. (2016) A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions. Mol. Microbiol., 99:909-924. 

Yu, Z., Armant, O. & Fischer, R. (2016) Fungi use the SakA/HogA pathway for phytochrome-dependent light signaling. Nature Microbiol., 2016, 1: 16019.

Yu, Z. & Fischer, R. (2019) Light sensing and responses in fungi. Nat. Rev. Microbiol., 17(1):25-36.

 

 

Dicovery of novel microtubule-organizing centers at septa

Microtubule organizing centers are essential in all eukaryotic cells. In higher eukaryotes the centrosome fullfills that function. In fungi the functional analogs of centrosomes are the MTOCs embedded into the nuclear envelope. They are called spindle-pole bodies, because during mitosis the SPB divides and make up the two poles of the spindle. Our discovery of additional MTOCs at septa was against the dogma that SPBs were the only MTOCs in fungi. The movie shows the dynamics of a MT-plus end associated motor protein, KipA. KipA-GFP appears comet-like and decorate the end of the MTs. The signals emerge clearly also from septa, which are indicated by the arrow heads.

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Scheme of MTOCs in A. nidulans

In 2003 the genome of Aspergillus nidulans was sequenced in a company in Boston, and we had access to the sequence for annotation before the sequence was published. We picked the kinesin motor proteins and started analyzing them one by one in the following years. One of the motors (KipA) localized to microtuble plus ends and could be used as plus-end tracking protein (like EB1 in higher eukaryotes). We noticed that microtubules were not only emanating from spindle-pole bodies but also from septa. A new microtubule-organizing center (MTOC) was discovered!

At the same time we characterized a novel protein in A. nidulans, ApsB, and found that it was a component of SPBs and of sMTOCs. This work was the basis for many years of research on MTOCs. We recently discovered that the composition of the different MTOCs in the cells is specific for certain MTOCs and that the composition is very dynamic.

Konzack, S., Rischitor, E. P., Enke, C. & Fischer, R. (2005) The role of the kinesin motor KipA in microtubule organization and polarized growth of Aspergillus nidulans. Mol. Biol. Cell, 16:497-506.

Veith, D., Scherr, N., Efimov, V.P. & Fischer, R. (2005) Role of the spindle-pole body protein ApsB and the cortex protein ApsA in microtubule organization and nuclear migration in Aspergillus nidulans. J. Cell Sci., 118:3705-3716. 

Zekert, N., Veith, D. & Fischer, R. (2010) Interaction of the Aspergillus nidulans MTOC component ApsB with gamma-tubulin and evidence for a role of a subclass of peroxisomes in the formation of septal MTOCs. Eukaryot. Cell, 9:795-805. 

Zhang, Y., Gao, X. Manck, R. Schmid, M., Osmani, A.H., Osmani, S.A., Takeshita, N. & Fischer, R. (2017) Microbutule-organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein. Mol. Microbiol., 106(2): 285-303.

Gao, X., Schmid, M., Zhang, Y., Fukuda, S., Takeshita, N. & Fischer, R. (2019) The spindle pole body of Aspergillus nidulans is asymmetrically composed and highly dynamic. J. Cell Sci.,132, 234799

Nuclear migration in Aspergillus nidulans

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Nuclei were labeled with GFP.

Soon after the discovery of GFP in 1994 and the heterologous expression in E. coli, we established the tool in Aspergillus nidulans. The first GFP version we tried did not work in A. nidulans, but then we received a new version from Regine Kahmanns lab in Munich and that worked well. We were able to target the GFP as a fusion protein to nuclei and taped first movies of nuclear migration in this fungus. We have distributed the plasmids and strain to many labs worldwide. 

The work on nuclear migration and the use of GFP was the many years of research on the cell biology of fungi.

Suelmann, R. & Fischer R. (1997) Nuclear traffic in fungal hyphae: In vivo study of nuclear migration and positioning in Aspergillus nidulans. Mol. Microbiol., 25(4), 757-769. (with cover)

Suelmann, R., Sievers, N., Galetzka, D., Robertson, L., Timberlake, W.E. & Fischer R. (1998) Nuclear traffic chaos in hyphae of apsB mutants of Aspergillus nidulans: Molecular characterization of apsB and in vivo observations of nuclear behaviour. Mol. Microbiol., 30(4), 831-842.

Krüger, M. & Fischer, R. (1998) Integrity of a Zn-finger like domain in SamB is crucial for morphogenesis in ascomycetous fungi. EMBO J., 17(1), 204-214.

Toews, M.W., Warmbold, J., Konzack, S., Rischitor, E.P., Veith, D., Vinuesa, C., Vienken, K., Wei, H. & Fischer, R. (2004) Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in vitro (GATEWAY). Curr. Genet., 45:883-889.

Rischitor, E.P., Konzack, S. & Fischer, R. (2004) The Kip3-like kinesin KipB moves along microtubules and determines spindle position during synchronized mitoses in Aspergillus nidulans hyphae. Eukaryot. Cell, 3(3):632-645.

Xiang, X. & Fischer, R. (2004) Nuclear migration and positioning in filamentous fungi. Fungal Genet. & Biol., 41, 411-419.