Recombinant Ashbya gossypii Probable kinetochore protein SPC25 (SPC25)

What is the function of SPC25 in Ashbya gossypii?

SPC25 forms part of the essential Ndc80 complex (consisting of Ndc80, Nuf2, Spc24, and Spc25), which serves as the key microtubule-binding element of the kinetochore. This complex is crucial for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to microtubule dynamics. The Spc24/Spc25 heterodimer specifically localizes to one end of the Ndc80 complex and binds to the kinetochore, while the N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex . This arrangement creates a force coupling system critical for chromosome segregation during cell division.

How conserved is SPC25 between A. gossypii and S. cerevisiae?

The high degree of genomic similarity between these species suggests significant conservation. More than 90% of A. gossypii genes show both homology and a particular pattern of synteny with Saccharomyces cerevisiae . The A. gossypii genome project was specifically initiated when conservation of gene order and orientation to S. cerevisiae was noted . For SPC25 specifically, conservation is evidenced by the functional interchangeability of many kinetochore components between these related fungi. Sequence comparison reveals that key functional domains, particularly those mediating protein-protein interactions within the kinetochore complex, are highly conserved, though species-specific differences exist that likely reflect adaptations to different growth patterns (filamentous versus budding).

What are the known interaction partners of A. gossypii SPC25?

SPC25 has several confirmed interaction partners within the kinetochore architecture:

The interaction with CENP-T appears particularly critical, as altering the Cnn1-binding site on Spc24-25 (e.g., through V159D mutation in Spc25) is lethal, suggesting this interaction is essential for proper kinetochore function .

What expression systems are suitable for recombinant A. gossypii SPC25?

Several expression systems have proven effective for recombinant expression of A. gossypii proteins:

• Homologous expression in A. gossypii: The fungus itself serves as an excellent host for recombinant protein expression. Recombinant proteins have been successfully expressed in A. gossypii from 2-mm plasmids, with expression levels often exceeding those in S. cerevisiae . For optimal expression, the AgTEF promoter has demonstrated superior performance, leading to 2- and 8-fold higher expression levels than the AgGPD promoter and heterologous promoters (ScADH1 and ScPGK1), respectively .

• Heterologous expression in bacteria: For structural and biochemical studies, bacterial expression systems have been used successfully for kinetochore components including the Ndc80 complex. E. coli-based expression allows for high-yield production of recombinant proteins for in vitro analyses .

• Alternative promoters: Recent research has identified several strong constitutive promoters for use in A. gossypii, including PCCW12, PTDH3, and PSED1, which can be selected based on desired expression levels .

For optimal purification results:

• Complex purification: Consider co-expressing SPC25 with its binding partner SPC24, as the stability of these proteins is often enhanced when expressed as a complex. The SPC24-SPC25 heterodimer can be expressed with a tag on one subunit for purification .

• Affinity chromatography: For tagged proteins, use appropriate affinity chromatography (e.g., Ni-NTA for His-tagged proteins, anti-myc for myc-tagged proteins).

• Buffer optimization: Include phosphatase inhibitors if studying phosphorylation-dependent interactions, as phosphorylation of interacting partners (e.g., CENP-T) can affect binding to the SPC24-SPC25 complex .

• Quality control: Verify proper folding through circular dichroism, limited proteolysis, and functional binding assays to known interaction partners.

How can I assess the functionality of recombinant A. gossypii SPC25?

Multiple approaches can verify the functionality of recombinant SPC25:

• Complementation studies: Test whether recombinant SPC25 can complement a deletion of the endogenous gene. This can be done by integrating the recombinant gene into the URA3 locus of a heterozygous diploid strain (SPC25/spc25Δ) and analyzing spore viability after sporulation .

• Protein-protein interaction assays: Perform in vitro binding assays between purified SPC25 (in complex with SPC24) and known binding partners such as Cnn1/CENP-T or the Mtw1 complex. Interaction can be assessed by analytical methods such as isothermal titration calorimetry, surface plasmon resonance, or pull-down assays .

• In vivo localization: Tag SPC25 with GFP and verify its proper localization to kinetochores during the cell cycle using fluorescence microscopy techniques similar to those described for other kinetochore proteins .

• FRET-based assays: Adapt FRET tension sensor approaches to measure interactions and conformational changes involving SPC25 within the kinetochore complex .

What microscopy techniques are most useful for studying A. gossypii SPC25 dynamics?

Given the unique multinucleate nature of A. gossypii, specialized microscopy approaches are needed:

• Time-lapse fluorescence microscopy: For GFP-tagged SPC25, use spinning disk confocal microscopy with a Plan-Apochromat 100x/1.49 NA oil-immersion objective to capture dynamic behavior across multiple nuclei . This approach has been successfully used to study the polarisome component AgSpa2p-GFP .

• Synchrony analysis: Although nuclei in A. gossypii divide asynchronously, you can analyze the degree of synchrony between adjacent nuclei using methods described in Langner et al. (2023) :

  • Fix cells with formaldehyde (3.7%)

  • Digest with zymolyase to remove the cell wall

  • Stain with anti-alpha Tubulin antibody and DAPI

  • Score cell cycle stages based on tubulin staining patterns

  • Calculate synchrony scores (observed versus expected pairs of nuclei in the same cell cycle stage)

• Fixed sample immunofluorescence: For colocalization studies, use antibodies against kinetochore markers in combination with anti-SPC25 antibodies. Commercial antibodies specific to A. gossypii SPC25 are available (e.g., CSB-PA751720XA01DOT) .

How can I study the role of SPC25 in kinetochore assembly in multinucleate A. gossypii?

The multinucleate context of A. gossypii presents unique opportunities to study kinetochore assembly:

• Conditional mutants: Generate temperature-sensitive alleles of SPC25 to study the immediate effects of protein inactivation on kinetochore structure and function.

• Nuclear autonomy analysis: Investigate whether perturbations to SPC25 affect all nuclei uniformly or create differential effects in a shared cytoplasm. This can be done using the synchrony analysis methods described above .

• Spatial organization: Examine whether SPC25 distribution correlates with sites of septin rings, as mitoses in A. gossypii are most common near cortical septin rings found at growing tips and branchpoints .

• Co-visualization approaches: Combine SPC25-GFP with markers for nuclear division cycle stages, such as tubulin (spindle visualization) and DAPI (DNA), to correlate SPC25 dynamics with specific stages of nuclear division .

How does A. gossypii SPC25 contribute to nuclear asynchrony in a shared cytoplasm?

A. gossypii presents an intriguing model where multiple nuclei divide asynchronously despite sharing a common cytoplasm. Research approaches to understand SPC25's potential role include:

• Local regulation hypothesis: Investigate whether SPC25 or its interactors are regulated locally around individual nuclei. The Whi3 protein forms biomolecular condensates near nuclei that contribute to asynchronous division . Examine whether SPC25 interacts with or is affected by these condensates.

• Phosphorylation analysis: Study whether CDK-dependent phosphorylation states of kinetochore components (including SPC25 interactors like CENP-T) vary between neighboring nuclei, potentially contributing to their asynchronous behavior .

• G1/S transition focus: Examine whether SPC25 influences the G1/S transition, which is a key point where sister nuclei begin to lose synchrony. The conserved G1 regulatory circuit involving Whi5 promotes asynchronous behavior , and interactions between this pathway and kinetochore assembly could be significant.

• Temperature-dependent analysis: Investigate whether SPC25 function varies with temperature, as A. gossypii isolates from different climate zones show different synchrony patterns at various temperatures .

How do mutations in SPC25 affect kinetochore tension and force coupling?

Understanding how SPC25 mutations affect force transmission at the kinetochore:

• FRET tension sensors: Adapt the FRET tension sensor approach described for Ndc80 to measure how SPC25 mutations affect force transmission across the kinetochore. This could involve inserting a FRET sensor into SPC25 or its interacting partners.

• Metaphase kinetochore mechanics: Use the mechanical model for the Ndc80 force coupler described in Suzuki et al. (2016) to predict how SPC25 mutations might affect force balance during metaphase.

• Binding interface mutations: Generate specific mutations at the SPC25-CENP-T interface (similar to the V159D mutation described in ) and analyze their effects on kinetochore assembly and tension generation.

• Comparison with other fungi: Compare the effects of equivalent mutations in SPC25 between A. gossypii and S. cerevisiae to identify potential differences in kinetochore mechanics between filamentous and budding yeast growth forms.

What is the relationship between polarized growth machinery and kinetochore function in A. gossypii?

A. gossypii exhibits rapid polarized hyphal growth, and exploring connections between polarization and chromosome segregation machinery provides insight into coordinated cellular processes:

• Co-localization studies: Investigate whether kinetochore components including SPC25 show spatial relationships with polarity determinants. The polarisome component AgSpa2p localizes to sites of polarized growth , while septins influence the spatial distribution of mitosis .

• Growth phase correlation: Compare SPC25 dynamics between the initial slow growth phase (6-10 μm/h) and mature hyphae (up to 200 μm/h) to determine if kinetochore assembly adapts to different growth modes.

• Formin interaction network: Explore potential functional links between formins like AgBni1 (essential for polarized growth and hyphal tip extension) and kinetochore components including SPC25.

• Nutrient signaling pathway: Investigate how starvation-induced signaling, which affects nuclear density through AgSwe1p-dependent CDK phosphorylation , influences kinetochore component assembly and function.

How can genome editing technologies be applied to study SPC25 function in A. gossypii?

Advanced genome editing approaches for SPC25 research:

• CRISPR/Cas9 and CRISPR-Cpf1: These systems have been recently adapted for multiplex genome editing in A. gossypii , enabling precise modifications to SPC25 and simultaneous editing of multiple kinetochore components.

• Domain swapping experiments: Use precise genome editing to replace domains of A. gossypii SPC25 with corresponding domains from other species to identify functionally divergent regions.

• Promoter engineering: Utilize the expanding toolbox of A. gossypii promoters (ranging from strong to weak expression) to create strains with controlled expression levels of SPC25 for dosage sensitivity studies.

• Tagging optimization: Combine CRISPR-based editing with the PCR-based gene targeting methods to create precisely tagged versions of SPC25 without disrupting native regulation.

Why is my recombinant A. gossypii SPC25 protein insoluble when expressed?

Insolubility often occurs because SPC25 naturally exists as part of a heterodimeric complex with SPC24. To resolve this issue:

• Co-expression strategy: Express SPC25 together with its binding partner SPC24, as the stability of these proteins is enhanced when expressed as a complex.

• Solubility tags: Incorporate solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO at the N-terminus.

• Expression conditions: Optimize expression conditions by reducing temperature (16-20°C), using slower induction with lower IPTG concentrations, or testing different media compositions.

• Denaturation-refolding: For severe cases, purify under denaturing conditions followed by controlled refolding in the presence of SPC24.

How can I distinguish between phenotypes caused by SPC25 mutation versus indirect effects?

To ensure observed phenotypes are directly attributable to SPC25:

• Complementation testing: Reintroduce wild-type SPC25 to verify phenotypic rescue.

• Domain-specific mutations: Create targeted mutations affecting specific interaction interfaces rather than complete gene deletions.

• Conditional alleles: Use temperature-sensitive or auxin-inducible degron approaches to enable acute inactivation, revealing immediate versus adaptive effects.

• Interaction partner analysis: Examine effects on known SPC25 binding partners (SPC24, CENP-T) to confirm the expected molecular consequences of your mutation.