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Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Aspergillus nidulans

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Cell Cycle Oscillators

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2329))

Abstract

Photoactivated localization microscopy (PALM), one of the super resolution microscopy methods improving the resolution limit to 20 nm, allows the detection of single molecules in complex protein structures in living cells. Microtubule-organizing centres (MTOCs) are large, multisubunit protein complexes, required for microtubule polymerization. The prominent MTOC in higher eukaryotes is the centrosome, and its functional ortholog in fungi is the spindle-pole body (SPB). There is ample evidence that besides centrosomes other MTOCs are important in eukaryotic cells. The filamentous ascomycetous fungus Aspergillus nidulans is a model organism, with hyphae consisting of multinucleate compartments separated by septa. In A. nidulans, besides the SPBs, a second type of MTOCs was discovered at septa (called septal MTOCs, sMTOC). All the MTOC components appear as big dots at SPBs and sMTOCs when tagged with a fluorescent protein and observed with conventional fluorescence microscopy due to the diffraction barrier. In this chapter, we describe the application of PALM in quantifying the numbers of individual proteins at both MTOC sites in A. nidulans and provide evidence that the composition of MTOCs is highly dynamic and dramatically changes during the cell cycle.

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Acknowledgments

Our research was in part financed by grants from the German Science Foundation (DFG Fi 459/20-1) and Japan Science and Technology Agency (JST, ERATO JPMJER1502).

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Authors and Affiliations

  1. Department of Microbiology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany

    Xiaolei Gao & Reinhard Fischer

  2. Microbiology Research Center for Sustainability (MiCS), Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan

    Norio Takeshita

Authors
  1. Xiaolei Gao
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  2. Reinhard Fischer
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  3. Norio Takeshita
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Corresponding author

Correspondence to Norio Takeshita .

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Editors and Affiliations

  1. School of Science and Technology, Nottingham Trent University, Nottingham, UK

    Amanda S. Coutts

  2. Scientific Writer, Newcastle upon Tyne, UK

    Louise Weston

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Gao, X., Fischer, R., Takeshita, N. (2021). Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Aspergillus nidulans . In: Coutts, A.S., Weston, L. (eds) Cell Cycle Oscillators . Methods in Molecular Biology, vol 2329. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1538-6_20

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  • DOI: https://doi.org/10.1007/978-1-0716-1538-6_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1537-9

  • Online ISBN: 978-1-0716-1538-6

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