Quantitative single molecule RNA-FISH and RNase-free cell wall digestion in Neurospora crassa

Abstract

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.

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.