Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa
Introduction
Neurospora crassa has been in use as a model organism for the study of many important biochemical, genetic, and cell biology processes in fungi and animals for nearly 100 years (Loros, 2020, Mela et al., 2020, Riquelme et al., 2011, Selker, 2013). There are several phenomena in Neurospora for which the visualization and quantification of mRNA molecules could help us better understand the mechanisms fungi and other syncytial cells use to regulate protein expression, cytoplasmic organization, and mRNA trafficking. mRNA profiling has revealed heterogeneous expression of mRNA throughout mycelia of N. crassa (Kasuga and Glass, 2008, Mela et al., 2020). For instance, differences in gene expression can be seen even at the level of individual nuclei depending on the local cytoplasmic environment (Pieuchot et al., 2015). In addition, many biological processes are likely impacted by post-transcriptional regulation including the circadian clock which appears to require highly complex post-transcriptional regulation of mRNA. This idea is supported by the fact there are temporal delays between peak mRNA accumulation and peak protein accumulation in genes regulated by the circadian clock, in addition to gene products that are rhythmically expressed at the level of mRNA or protein but not both (Hurley et al., 2014, Hurley et al., 2018). While tools to study mRNA regulation at the cellular level have been adapted and implemented for other filamentous fungi, none have been reported in Neurospora (Baumann et al., 2014, Lee et al., 2016).
Fluorescence imaging of intracellular molecules in fixed Neurospora hyphae is challenging. While there are examples in the literature, it is not commonplace and consensus methods for chemical fixation and permeabilization have not been established (Emerson et al., 2015, Managadze et al., 2010, Riquelme et al., 2002). A major obstacle for techniques that require the use of affinity probes in intact fixed fungal cells is the presence of a cell wall, which varies in composition throughout the fungal kingdom (Bourett et al., 1998, Imdahl and Saliba, 2020, Patel and Free, 2019). Additionally, we have found that mature, fixed Neurospora hyphae are recalcitrant toward adherence to glass or other surfaces commonly used for light microscopy. While there are several lytic enzyme preparations commercially available for permeabilization of fungal cell walls, they are not universally effective due to compositional differences in walls amongst fungi. Additionally, the enzymes tend to be harvested from cellular extracts that are enriched for fungal lytic enzyme activity by size or charge based chromatographic methods, but are not necessarily pure or free from contaminating nuclease activity, which is particularly important for the study of RNA.
Here we describe a method for liquid culture growth, chemical fixation, and digestion of the N. crassa cell wall in RNase-free conditions. RNase-free cell wall digestion employs a recombinant chitinase, for which we also provide a method for expression and purification. Finally, we have adapted single molecule RNA-FISH (smFISH) for use in Neurospora from protocols developed for other fungi (Lee et al., 2016, Raj et al., 2008). In total, this approach enables the quantitative analysis of mRNA localization and abundance in Neurospora opening up the ability to ask fundamental questions about RNA regulation in this key model system.
Section snippets
Recombinant Bacillus licheniformis chitinase as a tool for RNase-free cell wall digestion of N. crassa.
A critical first step in FISH protocols for fungi is permeabilizing the cell membrane and walls sufficiently to allow large, nucleic acid probes to enter the cell. The Neurospora cell wall is comprised of β-Glucans, chitin, galactomannan, and proteins. A compact layer of chitin is situated directly against the plasma membrane (Free, 2013, Verdín et al., 2019). Zymolyase-100T is a common enzymatic preparation used for digestion of cell walls in fungi (Lee et al., 2016, Li and Neuert, 2019).
Single molecule RNA-FISH in Neurospora crassa
smFISH is a powerful tool that has been validated and used in many systems to visualize mRNA in cells but underutilized in filamentous fungi. A major reason for this is likely the difficulty presented by cell wall permeabilization and maintaining cellular integrity throughout fixed-cell imaging procedures. Here, we present a protocol which was adapted from previous work in the filamentous fungus Ashbya gossypii (Dundon et al., 2016, Lee et al., 2016, Lee et al., 2013). Taken together, smFISH
Conclusions
Difficulties surrounding reliable fixation and permeabilization for fluorescence imaging methods has limited the application of these techniques in filamentous fungal research compared to more tractable systems like yeast, insects, and mammals. With recent advancements in single molecule imaging and super resolution microscopy techniques that require fixed cells, improved methods are necessary to enable biological discovery in filamentous fungi. The introduction of the recombinant chitinase
CRediT authorship contribution statement
Bradley M. Bartholomai: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Amy S. Gladfelter: Conceptualization, Methodology, Writing – review & editing, Supervision. Jennifer J. Loros: Conceptualization, Resources, Supervision, Project administration. Jay C. Dunlap: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors wish to thank Samantha Dundon, PhD for contributions to early work surrounding adaptation of the smFISH protocol for Neurospora. We thank Andreia Verissimo, PhD of Dartmouth’s BioMT core for considerable assistance in establishing a protocol for chitinase expression and purification. Additionally, we acknowledge the efforts of Zuzana Burdikova, PhD for her work in assisting with early attempts at chitinase purification.
Funding
This work was supported by the National Institutes of Health MIRA grant R35-GM118021 to Jay C. Dunlap; Dartmouth’s BioMT NIH NIGMS COBRE grant, P20-GM113132; and NIH training grant T32-008704 to Bradley M. Bartholomai.
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