Recombinant Neurospora crassa Probable kinetochore protein nuf-2 (nuf-2)

What is the molecular structure of Nuf-2 in Neurospora crassa?

Nuf-2 in N. crassa is a coiled-coil domain protein that forms part of the kinetochore complex. Similar to its yeast homolog, it contains regions with high potential for coiled-coil structure formation, which is critical for its interactions with other kinetochore proteins. The protein forms a heterodimer with NDC80, and together with SPC24 and SPC25, constitutes the complete NDC80 complex . Structural studies indicate the Ndc80-Nuf2 end folds into a dimeric arrangement of calponin-homology domains that contacts the microtubule surface through conserved positively charged residues .

How does Nuf-2 differ functionally between N. crassa and other model organisms?

While the core function of Nuf-2 in kinetochore assembly and chromosome segregation is conserved across species, recent research has revealed species-specific adaptations. In contrast to the yeast S. cerevisiae where Nuf2 is primarily associated with the spindle pole body and plays an essential role in nuclear division , the N. crassa Nuf-2 has recently been implicated in ribosome biogenesis processes beyond its canonical role in mitosis . This dual functionality represents an evolutionary adaptation specific to filamentous fungi. Unlike in mammalian cells where Nuf2 is preferentially modified by poly-SUMO-2/3 chains to facilitate CENP-E localization , the post-translational modification landscape of N. crassa Nuf-2 remains to be fully characterized.

What are the most effective methods for generating Nuf-2 knockout mutants in N. crassa?

The most effective approach for generating Nuf-2 knockout mutants in N. crassa utilizes strains deficient in non-homologous end-joining (NHEJ) DNA repair. Specifically, using strains with mutations in mus-51 or mus-52 (homologs of KU70 and KU80) significantly increases homologous recombination efficiency from ~10% to >90% . The methodology follows these steps:

• Design gene-specific primers with 29-nt common portions for the Nuf-2 gene using custom software tools .

• Generate a deletion cassette using yeast recombinational cloning, combining:

  • 5' and 3' flanking regions (~1 kb each) of the Nuf-2 gene

  • A hygromycin resistance cassette (hph)

  • A gapped yeast vector

• Transform the linear deletion cassette into N. crassa mus-51 or mus-52 mutant strains using electroporation.

• Select transformants on hygromycin-containing media.

• Confirm gene deletion by PCR and Southern blot analysis .

This approach achieves high-throughput gene disruption with an efficiency exceeding 90% in N. crassa .

What expression systems are optimal for producing recombinant Nuf-2 for structural and functional studies?

For structural and functional studies of recombinant N. crassa Nuf-2, heterologous expression in E. coli combined with the use of solubility tags has proven effective. The methodology should include:

• Codon optimization of the Nuf-2 sequence for the expression host, as N. crassa shows specific codon usage patterns .

• Generation of constructs encoding the full-length protein and domain-specific fragments, particularly the calponin homology domains and coiled-coil regions.

• Co-expression with binding partners (NDC80, SPC24, SPC25) to enhance stability and proper folding .

• Purification using affinity chromatography followed by size exclusion chromatography.

For functional studies requiring post-translational modifications, expression in insect cells using baculovirus systems may be more appropriate, especially when studying interactions that depend on specific modifications like SUMOylation .

How does Nuf-2 contribute to ribosome biogenesis in N. crassa?

Recent research has uncovered an unexpected role for Nuf-2 in ribosome biogenesis (RB) in addition to its canonical function in kinetochore assembly. According to studies published in 2025, siRNA depletion of NUF2 leads to:

• Reduced pre-rRNA transcription, the primary and rate-limiting step of ribosome biogenesis.

• Decreased levels of essential proteins for pre-rRNA transcription, including the largest subunit of RNA polymerase I (POLR1A) and RRN3.

• Reduced protein synthesis, likely due to decreased ribosome levels.

• Activation of the nucleolar stress pathway (NSP), evident by increased TP53 protein and CDKN1A (p21) mRNA levels .

These effects are also observed when other members of the NDC80 complex (NDC80, SPC24, SPC25) are depleted, indicating that the entire complex may contribute to this non-canonical function. This discovery establishes a novel connection between proteins involved in mitosis and nucleolar function during interphase .

What is the relationship between Nuf-2 and chromosome stability in N. crassa?

As a core component of the kinetochore complex, Nuf-2 plays a critical role in maintaining chromosome stability in N. crassa through:

• Formation of stable kinetochore-microtubule attachments during mitosis.

• Proper segregation of chromosomes during cell division.

• Potential involvement in centromere organization, as N. crassa centromeres comprise 175-300 kb of mutated degenerate transposons and AT-rich sequences .

Disruption of Nuf-2 function would likely result in chromosome missegregation, aneuploidy, and genomic instability. Studies of related proteins in N. crassa have shown that centromere and kinetochore defects can affect both mitotic and meiotic processes, potentially impacting the ordered arrangement of ascospores that reflects the strands of DNA participating in meiosis .

How do Nuf-2 protein levels depend on other kinetochore components in N. crassa?

Based on studies in other systems that are likely applicable to N. crassa, Nuf-2 protein levels show interdependence with other members of the NDC80 kinetochore complex (NDC80, SPC24, and SPC25). Research has demonstrated that:

• Depletion of NDC80, SPC24, or SPC25 results in reduced Nuf-2 levels starting at 24 hours post-depletion.

• This effect becomes more pronounced at 48 and 72 hours compared to negative controls.

• The co-dependency suggests that complex assembly is required for protein stability .

This interdependence has important implications for experimental design, as targeting any single component of the complex will likely affect the entire structure and associated functions.

What post-translational modifications regulate Nuf-2 function?

While specific post-translational modifications of N. crassa Nuf-2 have not been extensively characterized, research on homologous proteins in other organisms provides insights into likely regulatory mechanisms:

• SUMOylation: In mammalian cells, Nuf2 is preferentially modified by poly-SUMO-2/3 chains, which is upregulated during mitosis and correlates with CENP-E kinetochore localization .

• Potential phosphorylation sites: N. crassa possesses numerous kinases, including mitogen-activated protein kinases and cyclin-dependent kinases , which may regulate Nuf-2 activity through phosphorylation during the cell cycle.

• Complex formation-dependent stability: The interdependence of Nuf-2 levels with other NDC80 complex components suggests that proper complex assembly serves as a regulatory mechanism controlling Nuf-2 function .

For researchers studying these modifications, methodological approaches should include:

• Immunoprecipitation coupled with mass spectrometry

• Phospho-specific antibodies for western blotting

• SUMO-specific pulldown assays

• Site-directed mutagenesis of potential modification sites

How can genome editing techniques be optimized for studying Nuf-2 function in N. crassa?

For advanced genome editing of Nuf-2 in N. crassa, researchers should consider:

• CRISPR-Cas9 adaptation for N. crassa:

  • Design guide RNAs specific to the Nuf-2 locus

  • Optimize Cas9 expression using N. crassa-specific promoters

  • Deliver components using established transformation protocols

• Precise genetic modifications:

  • Create point mutations in functional domains using homology-directed repair

  • Insert epitope tags for protein tracking

  • Generate conditional alleles using inducible promoters

• Enhancing editing efficiency:

  • Use mus-51/mus-52 deletion strains to improve homologous recombination

  • Employ split-marker approaches for complex modifications

  • Implement fluorescence-based screening methods for rapid identification of successful edits

• Experimental validation:

  • Confirm edits by sequencing

  • Verify protein expression/modification by western blotting

  • Assess phenotypes during vegetative growth and sexual development

What approaches are effective for studying Nuf-2's dual role in kinetochore function and ribosome biogenesis?

To investigate the dual functionality of Nuf-2, researchers should employ:

• Domain-specific mutational analysis:

  • Generate truncations or point mutations that selectively impair either kinetochore or ribosomal functions

  • Assess each function using specific assays (spindle assembly for kinetochore function; pre-rRNA transcription for ribosome biogenesis)

• Cell cycle-specific studies:

  • Synchronize N. crassa cultures to examine Nuf-2 localization and interaction partners throughout the cell cycle

  • Use live-cell imaging with fluorescently tagged Nuf-2 to track its movement between the kinetochore and nucleolus

• Interaction network mapping:

  • Perform BioID or proximity labeling experiments to identify phase-specific interaction partners

  • Use chromatin immunoprecipitation to determine if Nuf-2 directly associates with rDNA

• Comparative analysis across species:

  • Determine if the dual functionality is conserved in other filamentous fungi

  • Express N. crassa Nuf-2 in S. cerevisiae to assess functional complementation

How should researchers interpret phenotypic data from Nuf-2 mutants in N. crassa?

When analyzing phenotypic data from Nuf-2 mutants in N. crassa, researchers should consider:

• Multiple phenotypic parameters:

  • Growth rate and morphology

  • Aerial hyphae formation and conidiation

  • Sexual development and ascospore production

  • Nuclear distribution and chromosome segregation

• Statistical approach:

  • Use appropriate statistical tests for quantitative phenotypes

  • Implement multiple biological and technical replicates

  • Consider environmental variables that may influence phenotypic expression

• Genetic background effects:

  • Compare phenotypes in different N. crassa strains (e.g., Oak Ridge vs. Mauriceville)

  • Assess interactions with other genetic factors, particularly genes involved in chromosome dynamics

• Distinguishing direct vs. indirect effects:

  • Use complementation with wild-type Nuf-2 to confirm phenotype causality

  • Implement rescue experiments with domain-specific constructs

  • Compare phenotypes with other kinetochore protein mutants

Based on previous studies of transcription factor knockouts in N. crassa, researchers should expect that approximately 43% of deletion mutants will show phenotypes, with more than half of these displaying multiple defects .

What bioinformatic tools are most useful for analyzing evolutionary conservation of Nuf-2 across fungal species?

For analyzing Nuf-2 conservation across fungal species, researchers should employ:

• Sequence analysis tools:

  • Multiple sequence alignment using MUSCLE or MAFFT

  • Profile hidden Markov models (HMMs) for sensitive homology detection

  • Phylogenetic tree construction using maximum likelihood methods

• Structural prediction and analysis:

  • Protein structure prediction using AlphaFold2

  • Coiled-coil prediction using COILS or Paircoil2

  • Domain identification using InterProScan

• Functional site prediction:

  • Conservation scoring to identify functionally important residues

  • Prediction of post-translational modification sites

  • Identification of protein-protein interaction interfaces

• Comparative genomics approaches:

  • Synteny analysis to examine genomic context

  • Assessment of codon usage patterns in different fungi

  • Analysis of gene neighborhood conservation

When interpreting results, researchers should note that a large proportion of N. crassa genes do not have homologs in yeasts like S. cerevisiae and S. pombe, with 57% of predicted proteins lacking good matches in these organisms . This evolutionary divergence highlights the importance of studying Nuf-2 in filamentous fungi specifically.

What strategies can address challenges in purifying functional recombinant Nuf-2 protein?

Researchers encountering difficulties with recombinant Nuf-2 purification should consider:

• Solubility enhancement:

  • Test multiple solubility tags (MBP, SUMO, Trx)

  • Optimize expression temperature (typically lower temperatures improve folding)

  • Include stabilizing additives in buffers (glycerol, low concentrations of detergents)

• Co-expression strategies:

  • Co-express with binding partners (NDC80, SPC24, SPC25)

  • Include chaperones to aid folding

  • Use bicistronic or polycistronic expression systems

• Alternative expression systems:

  • Switch from prokaryotic to eukaryotic expression

  • Test cell-free protein synthesis

  • Consider N. crassa-derived expression systems for native conditions

• Purification approach refinement:

  • Implement multi-step purification protocols

  • Use size exclusion chromatography as a final polishing step

  • Consider on-column refolding for challenging constructs

• Protein stability assessment:

  • Perform thermal shift assays to identify stabilizing buffer conditions

  • Conduct limited proteolysis to identify stable domains

  • Use dynamic light scattering to assess aggregation

How can researchers troubleshoot issues with gene targeting specificity when modifying Nuf-2 in N. crassa?

For addressing targeting specificity issues when modifying the Nuf-2 gene:

• Enhancing homologous recombination:

  • Use mus-51/mus-52 deletion strains to minimize non-homologous end joining

  • Extend the length of homology arms to 1.5-3 kb

  • Consider using split-marker approaches for challenging targets

• Improving construct design:

  • Verify target sequence against the updated N. crassa genome assembly

  • Check for repetitive elements or RIP-affected regions that may complicate targeting

  • Design primers with appropriate GC content and melting temperatures

• Optimizing transformation:

  • Adjust electroporation parameters for optimal DNA delivery

  • Use freshly prepared competent cells

  • Consider alternative transformation methods like biolistic bombardment

• Validation strategies:

  • Implement PCR-based screening with primers outside the targeted region

  • Perform Southern blot analysis to confirm single integration and absence of ectopic insertions

  • Sequence across integration junctions to verify precise modification

• Addressing common issues:

  • If RIP-affected regions are complicating targeting, design constructs to avoid these areas

  • For genes near centromeres or telomeres, adjust targeting strategies to account for chromatin structure

  • Consider temporary expression of Rad52 to enhance homologous recombination

What emerging technologies hold promise for advancing our understanding of Nuf-2 function?

Several cutting-edge technologies offer new avenues for investigating Nuf-2 function:

• Super-resolution microscopy:

  • Single-molecule localization microscopy (PALM/STORM) for precise localization studies

  • Structured illumination microscopy (SIM) for visualizing Nuf-2 dynamics during cell division

  • Expansion microscopy to better resolve kinetochore architecture

• Proteomics approaches:

  • Proximity-dependent biotinylation (BioID, TurboID) to map interaction networks

  • Crosslinking mass spectrometry to identify direct binding interfaces

  • Targeted proteomics for quantitative assessment of complex stoichiometry

• Genomics and epigenomics:

  • CUT&RUN or CUT&Tag for mapping Nuf-2 association with chromatin

  • Hi-C analysis to investigate potential roles in 3D genome organization

  • Multi-omics integration to correlate Nuf-2 function with transcriptional and translational outputs

• CRISPR-based technologies:

  • CRISPR interference/activation for tunable gene expression

  • Base editing for precise nucleotide substitutions

  • CRISPR-X for targeted mutagenesis of protein domains

• Computational approaches:

  • Molecular dynamics simulations to understand Nuf-2 conformational changes

  • Machine learning for predicting functional partners and regulatory networks

  • Systems biology modeling of kinetochore assembly and function

What are the most promising directions for therapeutic applications based on Nuf-2 research?

While the focus of this FAQ is on academic research rather than commercial applications, Nuf-2 research has several potential translational implications:

• Antifungal development:

  • Targeting fungal-specific aspects of Nuf-2 structure or function

  • Since many N. crassa genes lack homologs in yeasts , Nuf-2 may contain unique features that could serve as selective targets

• Cancer research applications:

  • Understanding kinetochore dysfunction in genomic instability

  • Parallels between Nuf-2's dual role in mitosis and ribosome biogenesis might inform cancer biology, as both processes are often dysregulated in cancer

• Cell cycle regulation insights:

  • Mechanisms connecting mitotic processes to interphase functions

  • Potential applications in addressing diseases with cell cycle defects

• Biotechnological applications:

  • Engineering N. crassa strains with modified Nuf-2 for enhanced protein production

  • Developing fungal biofactories with optimized growth and division properties

For researchers pursuing these directions, it will be essential to determine whether the dual functionality of Nuf-2 observed in N. crassa is conserved in pathogenic fungi and mammalian systems.

Table 1: Comparison of Nuf-2 Conservation and Functions Across Species

Table 2: Methods and Success Rates for Genetic Manipulation of N. crassa Genes Including Nuf-2

Table 3: Phenotypes Associated with Disruption of Kinetochore Proteins in N. crassa