Biology subjects

Gladfelter, A. S.

Publications and source records attributed to Gladfelter, A. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Life history stage-dependent nuclear selection in chimeric fungi

A single hyphal compartment of the filamentous fungus Neurospora crassa may contain tens or hundreds of nuclei, sharing macromolecules with each other, and, via a continuous cytoplasm, with the nuclei in other compartments. Nuclear lineages acquire mutations with each mitosis, which, combined with the autonomous mitosis of nuclei, has fueled speculation that multilevel selection may occur, both upon the mycelium, and upon individual nuclear populations. Here, we combine experiments on fungal chimera formed from two auxotrophically and epitopically labeled nuclear populations, with specially created microscopy toolkit for extracting the proportions of the two nucleotypes to analyze the strength of nuclear-level selective forces at different stages in the fungal life history. We find strong nucleotype-selective forces during spore-germination and establishment of the mycelium, and no evidence of selection on nuclei inside a growing mycelium. The kinetics of mycelial initiation from individual spores therefore allow for the selection of nuclear compositions best adapted to the fungus environment, in accordance with the hypothesized function of unicellular life history stages for purging deleterious mutations.

evolutionary biology↗

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2025

A phase oscillator model of cell cycles reveals nuclear density control in a branched fungal network

Multinucleate cells are widespread in biology, from human skeletal muscle and placenta to many filamentous fungi, yet their basic cell biology remains poorly understood. Maintaining an appropriate nuclear-to-cytoplasmic ratio is essential across cell types for physiological function, and mechanisms of size control have been extensively studied in mononucleate cells. Much less is known about how comparable control is achieved in cells where many nuclei share a common cytoplasm. The filamentous fungus Ashbya gossypii forms a branching mycelial network in which individual nuclei divide asynchronously, while the number of nuclei per cell volume (the nuclear density) is tightly controlled. How global regulation of nuclear density coexists with local cell cycle asynchrony remains unclear. We address this by combining a mathematical model of nuclear division in a growing and branching cell with live-cell microscopy. We model nuclei as a dividing population of phase oscillators within a branching cell network and parameterize the model with measurements from Ashbya cells. The model demonstrates that asynchrony is required to prevent large density fluctuations that would result from synchronous division, and that introducing a nuclear-density checkpoint to the cell cycles leads to synchrony if it is the only mechanism of density control. We find that coupling branch formation to nuclear density both stabilizes nuclear density and prevents the emergence of synchronous cycles. Our results indicate that asynchronous nuclear cycles together with density-responsive branching maintain a constant nuclear density, revealing a strategy for regulating the nuclear-to-cytoplasmic ratio in large multinucleate cells. SignificanceMultinucleate cells appear in diverse biological contexts, from human tissues to filamentous fungi, yet many fundamental aspects of their cell biology are still unclear. Regulating the nuclear-to-cytoplasmic ratio is important across cell types, and little is known about how this is achieved in large, multinucleate fungal cells. Using mathematical modeling and live-cell imaging, we identify how the density of nuclei is controlled in a growing and branching fungal mycelial network. We find that asynchronous nuclear cycles together with density-responsive branching can stabilize the nuclear-to-cytoplasmic ratio within a growing fungal network. This illustrates how large multinucleate cells can control nuclear density even as their morphology becomes increasingly complex.

cell biology↗

bioRxiv2025

mRNA interactions promote cotranslational association of heteromeric membrane proteins

Heteromeric membrane proteins play crucial physiological roles, yet how they are formed remains poorly understood. Heteromeric hERG1a/1b ion channels, essential for maintaining normal cardiac rhythm, assemble via cotranslational association of their encoding mRNAs. We hypothesized that direct hERG1a and 1b mRNA interactions facilitate this process. Using fluorescence colocalization and free energy of binding predictions, we found that hERG1a and 1b mRNAs form specific heterotypic condensates in vitro, suggesting direct interactions. When hERG1a mRNA was altered by synonymous mutations predicted to reduce its structural diversity and ability to interact with other mRNAs, overlap with hERG1b was dramatically diminished both in vitro and in cells, indicating weakened interactions. Reducing hERG1a structural diversity also influenced its translational complexes, defined by overlapping between fluorescently labeled mRNA and encoded protein (centroids within 400 nm). Whereas most of the wild-type hERG1a mRNA translates within heterotypic complexes, likely reflecting the biogenesis of hERG1a/1b heteromeric assemblies, reducing hERG1a structural diversity yielded more homotypic translational condensates and fewer hERG1a/1b heterotypic ones. This result suggests that the strength of mRNA interactions impact ion channel biogenesis. Further analysis of the heterotypic translational complexes revealed two distinct classes: a) simultaneous translation of both subunits and b) sequential association of fully translated hERG1b with translating hERG1a. Notably, reducing hERG1a structural diversity and interactions with 1b shifted translation toward the sequential mode. These findings identify a new role of mRNA sequence, structure, and interactions in orchestrating the cotranslational association of important heteromeric membrane proteins. SIGNIFICANCEThe physiological diversity of membrane protein complexes arises in part from the mixing of related yet functionally distinct subunits such as those forming "heteromeric" ion channels. How this mixing is achieved is not well understood. Here, we show that subunits composing heteromeric hERG1a/1b ion channels, crucial for normal cardiac function, predominantly associate in complexes where both subunits are simultaneously translated. This cotranslation is promoted by the association of the encoding mRNAs. Silent mutations disrupting hERG1a and 1b mRNA association, without altering the encoded protein, diminish simultaneous translation in favor of hERG1a translational complexes alone or in association with fully synthesized hERG1b. This study highlights the significant impact of RNA sequence, structure, and association on the formation of heteromeric membrane proteins.

biophysics↗

bioRxiv2025

Mechanisms of nuclear segregation in a multinucleate multibudding yeast

Budding yeasts present an especially challenging geometry for segregation of chromosomes, which must be delivered across the narrow mother-bud neck into the bud. Studies in the model yeast Saccharomyces cerevisiae have revealed an elaborate set of mechanisms that selectively orient one mitotic spindle pole towards the bud and then drive spindle elongation along the mother-bud axis, ensuring nuclear segregation between mother and bud. It is unclear how these pathways might be adapted to yield similar precision in more complex cell geometries. Here we provide the first description of the dynamics of mitosis in a multi-nucleate, multibudding yeast, Aureobasidium pullulans, and identify many unexpected differences from uninucleate yeasts. Mitotic spindles do not orient along the mother-bud axis prior to anaphase, and accurate nuclear segregation often occurs after spindle disassembly. Cortical Num1-dynein forces pull highly mobile nuclei into buds, and once a nucleus enters a bud, it discourages others from entering, ensuring that most daughters inherit only one nucleus.

cell biology↗

bioRxiv2025

Immiscible proteins compete for RNA binding to order condensate layers

Biomolecular condensates mediate diverse and essential cellular functions by compartmentalizing biochemical pathways. Many condensates have internal subdomains with distinct compositional identities. A major challenge lies in dissecting the multicomponent logic that relates biomolecular features to emergent condensate organization. Nuclear paraspeckles are paradigmatic examples of multi-domain condensates, comprising core and shell layers with distinct compositions that are scaffolded by the lncRNA NEAT1, which spans both layers. A prevailing model of paraspeckle assembly proposes that core proteins bind directly and specifically to core-associated NEAT1 domains. Combining informatics and biochemistry, we unexpectedly find that the essential core proteins FUS and NONO bind and condense preferentially with shell-associated NEAT1 domains. The shell protein TDP-43 exhibits similar NEAT1 domain preferences on its own but forms surfactant-like shell layers around core protein-driven condensates when both are present. Together, experiments and physics-based simulations suggest that competitive RNA binding and immiscibility between core and shell proteins orders paraspeckle layers. More generally, we propose that sub-condensate organization can spontaneously arise from a balance of collaborative and competitive protein binding to the same domains of a lncRNA. SignificanceThe cellular milieu is spatially organized into compartments called biomolecular condensates that exhibit rich internal organization which shapes their functions. Often comprising multiple proteins and RNAs, a major question concerns how molecular-scale features relate to emergent condensate forms. Here we study nuclear paraspeckles, archetypal multi-domain condensates comprising distinct core and shell layers assembled around a layer-spanning RNA scaffold. We find that different proteins associated with each layer all bind preferentially to the same shell-associated domains of the RNA scaffold. Core and shell proteins are inherently immiscible, establishing a competition that redirects core proteins to a suboptimal, core-associated domain of the scaffold. Our work reveals how a combination of competitive RNA binding and protein immiscibility can spatially organize multicomponent condensate subdomains.

biophysics↗

bioRxiv2025

Lipid packing and local geometry influence septin curvature sensing

Septins assemble into scaffolds that direct cell growth and morphology that are often localized to the plasma membrane. While septins preferentially bind convex membranes via amphipathic helices, their assembly on varied geometries in cells suggests additional localization cues. We tested the hypothesis that lipid composition directs septin assembly through lipid packing properties. Lipid mixtures varying in lipid packing were designed by molecular dynamics simulations and incorporated onto supported lipid bilayers to measure septin adsorption in vitro. Septins strongly favor loosely-packed, disordered lipid bilayers but additional geometry cues act in conjunction with this membrane property. Introducing tighter lipid packing in cells disrupted septin structures in a curvature dependent manner, specifically limiting septin assembly and retention along flat regions of the plasma membrane. This work demonstrates that packing defects and geometry jointly regulate septin localization and highlights how multiple membrane properties are integrated to control organization of the septin cytoskeleton. SummaryLocalization of the septin cytoskeleton is controlled by regulatory factors, membrane curvature, and charge. In this study, changes to lipid composition that modulate lipid packing defects are found to impact septin assemblies in vitro and in cells.

biochemistry↗

bioRxiv2025

Optimized vectors for genetic engineering of Aureobasidium pullulans

Aureobasidium pullulans is a polyextremotolerant black yeast that exhibits impressive morphological plasticity. Consequently, it shows promise as a model system for investigating mechanisms of cell adaptation to different environments and the regulation of cell shape. Here, we build upon the current toolkit for working with A. pullulans and design and test 25 vectors with seven different codon-optimized fluorophores and three selection cassettes. This includes vectors that allow for dual expression of GFP and mCherry tagged proteins at the URA3 locus and vectors that enable homology-based deletion or C-terminal tagging of endogenous genes without the need for cloning. This versatile vector series for working with A. pullulans will enable a broad range of experiments in this emerging model system. SUMMARYAn optimized plasmid toolkit for genetic engineering of the emerging model fungus, A. pullulans.

cell biology↗

bioRxiv2024

Negative feedback equalizes polarity sites in a multi-budding yeast

Morphogenesis in fungi and animals is directed by polarization of small GTPases Cdc42 and Rac. In the budding yeast Saccharomyces cerevisiae competition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeast Aureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1, and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.

cell biology↗

bioRxiv2024

Condensates act as translation hubs to coordinate multinucleate cell growth

Coordination between growth and nuclear division is a common cell feature. In some syncytia, nuclei divide asynchronously throughout the cell but growth occurs only at discrete locations, raising the question how the processes are locally regulated and globally coordinated. In the syncytial fungus Ashbya gossypii, both cell-cycle progression and hyphal elongation require condensates formed by the protein Whi3 in complex with distinct mRNA species. We show that Whi3 condensates are enriched for translation regulators and are associated with local, spatially patterned translation of specific target RNAs near nuclei and growth sites. Whi3-RNA condensates can both promote and repress mRNA translation in an RNA- and condensate size-dependent manner in vitro. Condensate-interfaces are sites of translation, tunable by condensate composition, RNA valency and protein charge-state in vitro. Together, these data suggest that Whi3 condensates can generate a continuum of translation states that vary depending on the subcellular location and resident RNA sequences.

cell biology↗

bioRxiv2024

RNA encodes physical information

Most amino acids are encoded by multiple codons, making the genetic code degenerate. Synonymous mutations affect protein translation and folding, but their impact on RNA itself is often neglected. We developed a genetic algorithm that introduces synonymous mutations to control the diversity of structures sampled by an mRNA. The behavior of the designed mRNAs reveals a physical code layered in the genetic code. We find that mRNA conformational heterogeneity directs physical properties and functional outputs of RNA-protein complexes and biomolecular condensates. The role of structure and disorder of proteins in biomolecular condensates is well appreciated, but we find that RNA conformational heterogeneity is equally important. This feature of RNA enables both evolution and engineers to build cellular structures with specific material and responsive properties.

biophysics↗

bioRxiv2024

Charge distribution and helical content tune the binding of septins amphipathic helix domain to lipid membranes

Septins are a class of cytoskeletal proteins that preferentially bind to domains of micron-scale curvature on the cell membrane. Studies have shown that amphipathic helix (AH) domains in septin oligomers are essential for septin curvature sensing. Yet, the underlying mechanochemical interactions that modulate this curvature sensing remain ambiguous. Here we use all-atom molecular dynamics alongside a metadynamics enhanced sampling approach to bridge the gap between time and length scales required to optimize and validate experimental design of amphipathic helices. Simulations revealed that the local charge on the termini of an 18-amino-acid AH peptide impacts its helical content and positioning within lipid membranes. These computational observations are confirmed with experiments measuring the binding of synthetic AH constructs with variable helical content and charged termini to lipid vesicles. Taken together, these results identify the helical content of amphipathic helices as a regulator of septin binding affinity to lipid membranes. Additionally, we examined an extended AH sequence including 8 amino acids upstream and downstream of the minimal 18-amino-acid-long AH domain to more closely mimic the native protein in simulations and experiments. Simulations and experiments show that the extended peptide sequence adopts a strong alpha-helical conformation when free in solution, giving rise to a higher affinity to lipid membranes than that of the shorter AH sequence. Together, these results provide insight into how the native septin proteins interact with membranes, and establish general design principles that can guide the interaction of future synthetic materials with lipid membranes in a programmable manner. STATEMENT OF SIGNIFICANCEUnderstanding how cells sense and react to their shape is necessary for numerous biological processes. Here we explore the interactions between amphipathic helices, a curvature sensing protein motif, and lipid membranes. Using molecular dynamics simulations, enhanced simulation sampling techniques, and experiments, we find that increasing the helical content of the amphipathic helix or adding charged capping sequences yields higher membrane binding affinity. Understanding these parameters for membrane-binding could enable us to interface and regulate native protein functions, as well as guide the design of synthetic curvature-sensing materials that can interact with and deform lipid membranes.

biophysics↗

bioRxiv2024

Comparative analysis of the syncytiotrophoblast in placenta tissue and trophoblast organoids using snRNA sequencing

The outer surface of chorionic villi in the human placenta consists of a single multinucleated cell called the syncytiotrophoblast (STB). The unique cellular ultrastructure of the STB presents challenges in deciphering its gene expression signature at the single-cell level, as the STB contains billions of nuclei in a single cell. There are many gaps in understanding the molecular mechanisms and developmental trajectories involved in STB formation and differentiation. To identify the underlying control of the STB, we performed comparative single nucleus (SN) and single cell (SC) RNA sequencing on placental tissue and tissue-derived trophoblast organoids (TOs). We found that SN RNA sequencing was essential to capture the STB population from both tissue and TOs. Differential gene expression and pseudotime analysis of TO-derived STB identified three distinct nuclear subtypes reminiscent of those recently identified in vivo. These included a juvenile nuclear population that exhibited both CTB and STB marker expression, a population enriched in genes involved in oxygen sensing, and finally a subtype enriched in transport and GTPase signaling molecules. Notably, suspension culture conditions of TOs that restore the native orientation of the STB (STBout) showed elevated expression of canonical STB markers and pregnancy hormones, along with a greater proportion of the STB nucleus subtype specializing in transport and GTPase signaling, compared to those cultivated with an inverted STB polarity (STBin). Gene regulatory analysis identified novel markers of STB differentiation conserved in tissue and TOs, including the chromatin remodeler RYBP, that exhibited STB-specific RNA and protein expression. CRISPR/Cas9 knockout of RYBP in STBin TOs did not impact cell-cell fusion; however, bulk RNA sequencing revealed downregulation of the pregnancy hormone CSH1 and upregulation of multiple genes associated with the oxygen-sensing STB nuclear subtype. Finally, we compared STB gene expression signatures amongst first trimester tissue, full-term tissue, and TOs, identifying many commonalities but also notable variability across each sample type. This indicates that STB gene expression is responsive to its environmental context. Our findings emphasize the utility of TOs to accurately model STB differentiation and the distinct nuclear subtypes observed in vivo, offering a versatile platform for unraveling the molecular mechanisms governing STB functions in placental biology and disease.

developmental biology↗

bioRxiv2024

A single septin from a polyextremotolerant yeast recapitulates many canonical functions of septin hetero-oligomers.

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septins homologs are conserved from yeasts to humans, the two systems in which septins have been studied most extensively. But there is also a fifth family of septin proteins that remain biochemically mysterious. Members of this family, known as Group 5 septins, appear in the genomes of filamentous fungi, and thus have been understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can serve as a model system for understudied Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola, demonstrating that this septin--by itself in vitro-- recapitulates many of the functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, senses membrane curvature, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher order structures formed by canonical septins, which in K. petricola cells form extended filaments. These findings provide insight into how septin hetero-oligomers evolved from ancient homomers and raise the possibility that Group 5 septins govern the higher order structures formed by canonical septins. Significance StatementO_LISeptins are understudied cytoskeletal proteins. Here, we biochemically characterized KpAspE, a novel Group 5 septin from a polyextremotolerant black fungus. C_LIO_LIKpAspE in isolation recapitulates many properties of canonical septin hetero-octamers in vitro and interacts with the Cdc11, the terminal subunit of those octamers. C_LIO_LIThese findings provide insight into how ancient septins may have evolved and diversified from homopolymers and suggest that many of the septin functions were present in the ancestral protein. C_LI

biochemistry↗

bioRxiv2023

Biomolecular condensates in fungi are tuned to function at specific temperatures

Temperature can impact every reaction and molecular interaction essential to a cell. For organisms that cannot regulate their own temperature, a major challenge is how to adapt to temperatures that fluctuate unpredictability and on variable timescales. Biomolecular condensation offers a possible mechanism for encoding temperature-responsiveness and robustness into cell biochemistry and organization. To explore this idea, we examined temperature adaptation in a filamentous-growing fungus called Ashbya gossypii that engages biomolecular condensates containing the RNA-binding protein Whi3 to regulate mitosis and morphogenesis. We collected wild isolates of Ashbya that originate in different climates and found that mitotic asynchrony and polarized growth, which are known to be controlled by the condensation of Whi3, are temperature sensitive. Sequence analysis in the wild strains revealed changes to specific domains within Whi3 known to be important in condensate formation. Using an in vitro condensate reconstitution assay we found that temperature impacts the relative abundance of protein to RNA within condensates and that this directly impacts the material properties of the droplets. Finally, we found that exchanging Whi3 genes between warm and cold isolates was sufficient to rescue some, but not all, condensate-related phenotypes. Together these data demonstrate that material properties of Whi3 condensates are temperature sensitive, that these properties are important for function, and that sequence optimizes properties for a given climate.

cell biology↗

bioRxiv2023

Dynamical control enables the formation of demixed biomolecular condensates

Macromolecular phase separation underlies the regulated formation and dissolution of biomolecular condensates. What is unclear is how condensates of distinct and shared macromolecular compositions form and coexist within cellular milieus. Here, we use theory and computation to establish thermodynamic criteria that must be satisfied to achieve compositionally distinct condensates. We applied these criteria to an archetypal ribonucleoprotein condensate and discovered that demixing into distinct protein-RNA condensates cannot be the result of purely thermodynamic considerations. Instead, demixed, compositionally distinct condensates arise due to asynchronies in timescales that emerge from differences in long-lived protein-RNA and RNA-RNA crosslinks. This type of dynamical control is also found to be active in live cells whereby asynchronous production of molecules is required for realizing demixed protein-RNA condensates. We find that interactions that exert dynamical control provide a versatile and generalizable way to influence the compositions of coexisting condensates in live cells.

biophysics↗

bioRxiv2022

A kinetic basis for curvature sensing by septins

The ability of cells to sense and communicate their shape is central to many of their functions. Much is known about how cells generate complex shapes, yet how they sense and respond to geometric cues remains poorly understood. Septins are GTP-binding proteins that localize to sites of micron-scale membrane curvature. Assembly of septins is a multi-step and multi-scale process but it is unknown how these discrete steps lead to curvature sensing. Here we experimentally examine the time-dependent binding of septins at different curvatures and septin bulk concentrations. These experiments unexpectedly indicated that the curvature preference of septins is not absolute but rather is sensitive to the combinations of membrane curvatures present in a reaction, suggesting there is competition between different curvatures for septin binding. To understand the basis of this result, we developed a kinetic model that connects septins self-assembly and curvature sensing properties. Our experimental and modeling results are consistent with curvature-sensitive assembly being driven by cooperative associations of septin oligomers in solution with the bound septins. When combined, the work indicates septin curvature sensing is kinetically determined, sensitive to bulk concentration, and the available membrane curvatures. While much geometry-sensitive assembly in biology is thought to be guided by intrinsic material properties of molecules, this is an important example of how kinetics can drive mesoscale curvature-sensitive assembly of polymers. Significance StatementCells use their membrane curvature to coordinate the activation and spatiotemporal compartmentalization of molecules during key cellular processes. Recent works have identified different proteins that can sense or induce membrane curvature from nano- to micron-scale. Septins are nanoscopic cytoskeletal proteins that preferentially bind to membranes with a narrow range of micron-scale curvatures. Yet the sensing mechanism remains ambiguous. Using a combination of microscopy and kinetic modeling, we show that, unlike most proteins that sense curvature in a single protein scale, curvature sensing in septins is determined kinetically through their multi-step hierarchical assembly on the membrane. This introduces a novel kinetic basis of fidelity, where the same protein can be deployed for differential binding sensitivities in different cellular contexts.

biophysics↗

bioRxiv2021

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

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.

cell biology↗