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Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution
Published on: June 23, 2022
Imaging Septum Formation by Fluorescence Microscopy
Juan Carlos Ribas1, Juan Carlos G Cortés2
1Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad de Salamanca, C/ Zacarias Gonzalez 1, 37007, Salamanca, Spain.
Abstract:
Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosin-based ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flanked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fission yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifically stained by the fluorochrome Calcofluor white. Recent findings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental approaches are needed. Here we describe the methods developed to image the septum structure by fluorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy.
Insights
This study details methods for visualizing fungal cell division septa using fluorescence microscopy. Researchers developed new techniques to observe septum formation and progression in Schizosaccharomyces pombe, aiding the study of cytokinesis.
Area of Science:
- Cell Biology
- Mycology
- Microscopy
Background:
- Fungal cytokinesis involves actomyosin ring contraction and septum formation.
- The fission yeast Schizosaccharomyces pombe forms a unique β(1,3)glucan-rich primary septum.
- Septum synthesis is crucial for proper ring contraction during cell division.
Purpose of the Study:
- To develop and describe methods for imaging fungal septum structure.
- To analyze septum progression during cytokinesis using time-lapse microscopy.
- To investigate the molecular links between the septum and cytokinetic machinery.
Main Methods:
- Transmission electron microscopy (TEM) for detailed septum structure analysis.
- Fluorescence microscopy for visualizing septum formation and progression.
- Time-lapse microscopy to observe dynamic changes during cytokinesis.
Main Results:
- The study describes established methods for fluorescence imaging of fungal septa.
- Time-lapse microscopy allows for detailed analysis of septum progression.
- These methods facilitate the study of molecular mechanisms connecting septum synthesis to cytokinesis.
Conclusions:
- New fluorescence microscopy methods are presented for studying fungal cell division.
- The techniques enable detailed observation of septum formation and its role in cytokinesis.
- Further research can utilize these methods to explore the molecular basis of fungal cytokinesis.
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