Recombinant Ashbya gossypii Monopolar spindle protein 2 (MPS2)

What is Ashbya gossypii Monopolar Spindle Protein 2 (MPS2) and what is its significance in fungal biology?

MPS2 (UniProt ID: Q755A9) is a 338-amino acid protein found in the filamentous fungus Ashbya gossypii. The protein is believed to play important roles in spindle pole body functions and potentially in the establishment and maintenance of polarized growth, which is characteristic of filamentous fungi. Understanding MPS2 function contributes to broader knowledge of fungal developmental biology, particularly in the context of differences between filamentous growth and yeast growth patterns. A. gossypii serves as an excellent model organism to study these processes as it is genetically tractable and has a completed genome sequence . The study of MPS2 may provide insights relevant to other fungal species, including dimorphic pathogens like Candida albicans, where morphological transitions are linked to virulence .

What expression systems are commonly used for recombinant A. gossypii MPS2 production?

E. coli is the most documented expression system for recombinant A. gossypii MPS2 production. The bacterial expression system allows for efficient production of the full-length protein (1-338 aa) with fusion tags such as the N-terminal His tag, which facilitates subsequent purification steps . The expression in E. coli offers advantages including high yield, scalability, and well-established protocols for protein induction and extraction. Researchers should consider optimizing codon usage for efficient expression in the bacterial host, as fungal codons may not be optimal for E. coli translation machinery.

What are the recommended storage and handling conditions for recombinant MPS2 protein?

For optimal stability and activity maintenance of recombinant A. gossypii MPS2, the following storage and handling conditions are recommended:

• Store the lyophilized protein powder at -20°C/-80°C upon receipt

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• For reconstituted protein with 50% glycerol, store at -20°C/-80°C

• Avoid repeated freezing and thawing which can compromise protein integrity

How can researchers design experiments to investigate MPS2's potential role in hyphal growth and polarity establishment?

To investigate MPS2's role in hyphal growth and polarity establishment, researchers should consider a multi-faceted experimental approach:

• Gene deletion and mutational analysis: Create Agmps2Δ strains and observe phenotypic changes in hyphal growth patterns, similar to approaches used for studying other polarity factors like AgAxl2 and AgBoi1/2 . Focus on potential defects in hyphal tip growth, branch formation, and responses to polarity cues.

• Fluorescent protein tagging: Generate GFP or mCherry fusions of MPS2 to monitor its localization during hyphal growth, particularly at hyphal tips, branch points, and septin rings, which are critical for polarity maintenance in A. gossypii .

• Co-localization studies: Examine potential interactions with known polarity factors such as AgBoi1/2, which has been shown to collaborate with AgRho3 to prevent nonpolar growth at hyphal tips . Additionally, investigate co-localization with actin patches and cables, as their distribution reflects polarity maintenance status.

• Stress response assays: Test Agmps2Δ strains under various stress conditions (heat, osmotic, cell wall stresses) to assess whether MPS2 integrates polarity establishment with environmental responses, similar to what has been observed with AgAxl2 .

• Time-lapse microscopy: Monitor dynamics of hyphal tip growth in wild-type versus Agmps2Δ strains to identify potential defects in sustained polarized growth, focusing on parameters such as growth rate, directionality, and tip morphology.

What promoter systems would be optimal for controlled expression studies of MPS2 in A. gossypii?

For controlled expression studies of MPS2 in A. gossypii, researchers should select promoters based on specific experimental requirements:

For precise measurement of promoter activity, researchers can adapt the Dual Luciferase Reporter (DLR) Assay that has been validated for A. gossypii . Since episomic vectors are not fully stable in A. gossypii, genomic integrative cassettes should be used for consistent expression . This approach would allow for quantitative assessment of MPS2 expression levels under different promoters and conditions.

How might MPS2 function in relation to other polarity factors in A. gossypii?

MPS2 likely functions within a complex network of polarity factors in A. gossypii. Based on studies of related polarity proteins, we can hypothesize several potential relationships:

• Interaction with Rho GTPases: MPS2 may function alongside Rho-type GTPases such as AgRho3, which is known to prevent nonpolar growth at hyphal tips . This interaction could be part of a signaling cascade that maintains polarized growth.

• Association with Boi proteins: AgBoi1/2, an SH3/PH domain protein that localizes to hyphal tips, is required for preventing nonpolar growth . MPS2 could function in parallel or in concert with this pathway to maintain proper hyphal morphology.

• Integration with Axl2 pathways: AgAxl2 integrates polarity establishment, maintenance, and environmental stress response in A. gossypii . MPS2 might contribute to this integrative function, especially in contexts where nuclear positioning and spindle orientation are coordinated with polarized growth.

• Connection to actin cytoskeleton: Since polarity maintenance in A. gossypii involves proper organization of actin patches and cables , MPS2 could play a role in regulating actin dynamics, potentially through interactions with actin nucleation or stabilizing factors.

Unlike the transient polarization seen in budding yeast, A. gossypii maintains sites of polarity for extended periods of growth while establishing new polarity sites . MPS2 might be specifically adapted to this sustained polarity maintenance that characterizes filamentous growth.

What methodological approaches are recommended for creating MPS2 mutants in A. gossypii?

For creating MPS2 mutants in A. gossypii, researchers should consider the following methodological approaches:

• PCR-based gene targeting: Utilize PCR-generated deletion cassettes containing selectable markers (such as loxP-KanMX-loxP) flanked by homologous regions targeting the MPS2 locus . The ease of genetic manipulation in A. gossypii makes this approach particularly effective.

• Domain-specific mutations: Design mutations targeting specific functional domains of MPS2 based on sequence analysis and structural predictions. This approach can help dissect domain-specific functions without completely eliminating the protein.

• Integration of expression cassettes: For controlled expression studies, integrate cassettes containing MPS2 variants under different promoters (as described in 2.2) at neutral genomic loci using recombinogenic flanks that target sites such as ADR304W or AGL034C .

• Cre-loxP system: Implement the Cre-loxP system for marker recycling when creating multiple mutations or for marker removal after successful integration .

• Verification protocols: Confirm successful mutations through:

  • PCR verification of correct integration

  • Southern blotting for complex modifications

  • Sequencing to verify sequence integrity

  • Western blotting to confirm protein expression levels or absence

  • Phenotypic analysis focusing on hyphal growth patterns and polarity maintenance

How does MPS2 function compare between A. gossypii and related yeast species?

A comparative analysis of MPS2 function between A. gossypii and related yeast species reveals important evolutionary and functional insights:

• Morphological context: While S. cerevisiae alternates between polarized and isotropic growth during its cell cycle, A. gossypii exhibits persistent highly polarized growth with multiple axes of polarity coexisting in a single cell . This fundamental difference likely influences MPS2 function in the context of spindle pole body dynamics and nuclear division.

• Functional adaptation: In filamentous fungi like A. gossypii, MPS2 may have evolved specialized functions to coordinate nuclear division with the continuous hyphal extension, whereas in S. cerevisiae, its functions are adapted to the budding cell cycle.

• Polarity maintenance: Unlike S. cerevisiae where polarity establishment is transient and cyclical, A. gossypii maintains polarity for extended periods . MPS2 may contribute to this sustained polarity, potentially through interactions with filamentous-specific factors.

• Stress response integration: The integration of polarity with stress response pathways appears to be a common feature in fungi, as seen with the AgAxl2 protein . MPS2 might similarly participate in integrating nuclear division events with environmental responses in the filamentous context.

• Relevance to dimorphic fungi: Understanding MPS2 function in A. gossypii may provide insights into morphological transitions in dimorphic pathogens like C. albicans, where switching between yeast and filamentous forms is critical for virulence .

What are the optimal purification methods for recombinant A. gossypii MPS2 protein?

For optimal purification of His-tagged recombinant A. gossypii MPS2 protein from E. coli expression systems, the following protocol is recommended:

• Cell lysis:

  • Resuspend bacterial pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, protease inhibitor cocktail)

  • Lyse cells by sonication or French press

  • Clarify lysate by centrifugation at 15,000 × g for 30 minutes at 4°C

• Affinity chromatography:

  • Load clarified lysate onto Ni-NTA resin equilibrated with binding buffer

  • Wash extensively with wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)

  • Elute protein with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole)

• Buffer exchange and concentration:

  • Pool fractions containing MPS2 (verify by SDS-PAGE)

  • Exchange buffer to storage buffer (Tris/PBS-based buffer, pH 8.0) using dialysis or desalting columns

  • Add 6% trehalose as a stabilizing agent

• Quality control:

  • Verify purity by SDS-PAGE (>90% purity is expected)

  • Confirm identity by Western blotting or mass spectrometry

  • Assess protein activity through appropriate functional assays

• Storage:

  • Lyophilize the purified protein or prepare aliquots with 5-50% glycerol

  • Store at -20°C/-80°C for long-term preservation

How can researchers assess the functional integrity of purified recombinant MPS2?

To assess the functional integrity of purified recombinant A. gossypii MPS2 protein, researchers should consider multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Thermal shift assays to determine protein stability

  • Size exclusion chromatography to assess oligomeric state

• Binding activity assays:

  • Pull-down assays to identify potential binding partners from A. gossypii extracts

  • Surface plasmon resonance (SPR) to measure binding kinetics with known or predicted interactors

  • Microscale thermophoresis (MST) for detecting interactions with small molecules or proteins

• Functional reconstitution:

  • In vitro assays that reconstitute aspects of spindle pole body assembly or function

  • Microtubule binding/polymerization assays if MPS2 interacts with microtubules

  • Complementation assays using purified protein introduced into mps2Δ cells

• Identification of post-translational modifications:

  • Mass spectrometry analysis to identify potential phosphorylation or other modifications

  • Phosphatase treatment to assess the impact of phosphorylation on protein function

  • Site-directed mutagenesis of potential modification sites followed by functional assays

• Immunological detection:

  • Development of specific antibodies against MPS2 for detection and localization studies

  • Immunoprecipitation to validate protein-protein interactions

What approaches can be used to study the role of MPS2 in nuclear division and organization in A. gossypii?

To investigate MPS2's role in nuclear division and organization in A. gossypii, researchers should implement these complementary approaches:

• Live-cell imaging:

  • Generate MPS2-fluorescent protein fusions for real-time localization studies

  • Use nuclear markers (e.g., histone-GFP) to track nuclear position and division

  • Employ time-lapse microscopy to monitor nuclear dynamics during hyphal growth

• Genetic manipulation:

  • Create conditional MPS2 mutants using regulatable promoters like PHSP26

  • Design temperature-sensitive alleles to allow rapid inactivation of MPS2 function

  • Implement CRISPR/Cas9 genome editing for precise modification of MPS2 domains

• Spindle pole body (SPB) analysis:

  • Use electron microscopy to examine SPB structure in wild-type versus MPS2 mutants

  • Perform immunofluorescence microscopy with SPB markers to assess MPS2's impact on SPB duplication and separation

  • Analyze nuclear division defects resulting from MPS2 dysfunction

• Nuclear positioning:

  • Examine potential connections between MPS2 and cytoskeletal elements (actin, microtubules) that position nuclei in hyphae

  • Investigate coordination between nuclear division and hyphal growth/branching in MPS2 mutants

  • Study the impact of environmental stresses on nuclear organization in wild-type versus MPS2 mutant strains

• Coordination with polarity factors:

  • Investigate potential interactions between MPS2 and known polarity factors such as AgBoi1/2 and AgAxl2

  • Examine whether polarity defects in MPS2 mutants are primary or secondary to nuclear division defects

  • Study how MPS2 might integrate nuclear positioning with polarized growth in the multinucleate hyphae of A. gossypii

How can heterologous expression systems be optimized for high-yield production of functional MPS2?

To optimize heterologous expression systems for high-yield production of functional A. gossypii MPS2, researchers should consider these strategies:

• Expression host selection:

  • E. coli is the established system for MPS2 expression , but consider:

    • BL21(DE3) strains for high-level expression

    • Rosetta strains for rare codon optimization

    • SHuffle strains for improved disulfide bond formation

  • Consider eukaryotic expression systems (e.g., P. pastoris) for complex folding or post-translational modifications

• Construct optimization:

  • Optimize codon usage for the selected expression host

  • Design constructs with various fusion tags (His, GST, MBP) to improve solubility

  • Create domain truncations if full-length protein presents expression challenges

  • Include precision protease sites for tag removal without affecting protein function

• Expression conditions:

  • Screen multiple induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Test various inducer concentrations and induction times

  • Evaluate the impact of additives such as:

    • Osmolytes (sorbitol, trehalose)

    • Folding chaperones (co-expression of GroEL/ES)

    • Mild detergents for membrane-associated domains

• Purification optimization:

  • Implement multi-step purification protocols including:

    • Affinity chromatography (Ni-NTA for His-tagged MPS2)

    • Ion exchange chromatography to separate charge variants

    • Size exclusion chromatography for final polishing and buffer exchange

  • Optimize buffer compositions to enhance stability (adding trehalose as used in commercial preparations)

• Scale-up considerations:

  • Develop fed-batch fermentation protocols for high-density cultures

  • Optimize oxygen transfer for aerobic expression

  • Implement automated purification strategies for consistency

How can MPS2 be utilized as a tool for studying evolutionary aspects of fungal morphogenesis?

MPS2 offers valuable opportunities for studying evolutionary aspects of fungal morphogenesis:

• Comparative genomics approach:

  • Compare MPS2 sequences across fungal lineages ranging from unicellular yeasts to filamentous fungi

  • Identify conserved domains versus lineage-specific adaptations that correlate with morphological complexity

  • Reconstruct the evolutionary history of MPS2 in relation to the emergence of filamentous growth

• Functional complementation studies:

  • Test whether MPS2 orthologs from different fungi can complement each other's functions

  • Determine which domains are responsible for species-specific functions

  • Create chimeric proteins to pinpoint regions that confer filamentous-specific versus yeast-specific functions

• Morphological transition models:

  • Utilize A. gossypii as a model to understand the role of MPS2 in filamentous growth

  • Compare with the dimorphic fungus C. albicans to assess MPS2's potential role in morphological transitions

  • Investigate whether MPS2 functions differ between constitutively filamentous fungi and those capable of dimorphic switching

• Molecular evolution analysis:

  • Assess selection pressures on different MPS2 domains across fungal lineages

  • Identify correlation between MPS2 sequence evolution and changes in fungal growth patterns

  • Study potential co-evolution with interacting partners involved in polarity and nuclear organization

• Ancestral reconstruction:

  • Use phylogenetic approaches to reconstruct ancestral MPS2 sequences

  • Test functionality of reconstructed ancestral proteins in modern fungi

  • Gain insights into the evolutionary trajectory of nuclear organization mechanisms in fungi

What are the key considerations when designing experiments to investigate MPS2 involvement in stress responses?

When investigating MPS2's potential involvement in stress responses, researchers should consider these experimental design elements:

• Stress condition selection:

  • Test multiple stress types: heat, osmotic, oxidative, cell wall, nutrient limitation

  • Establish dose-response relationships for each stress condition

  • Include both acute and chronic stress exposure protocols

  • Draw parallels with known stress-responsive proteins like AgAxl2

• Phenotypic analysis:

  • Compare growth rates and morphology of wild-type versus mps2Δ strains under stress

  • Examine nuclear organization and division during stress exposure

  • Analyze potential changes in MPS2 localization in response to different stresses

  • Assess recovery capabilities after stress removal

• Transcriptional regulation:

  • Use RT-qPCR to measure MPS2 expression changes under various stress conditions

  • Implement reporter gene assays using the MPS2 promoter

  • Compare with known stress-responsive genes to identify shared regulatory elements

  • Consider utilizing the Dual Luciferase Reporter system adapted for A. gossypii

• Genetic interaction studies:

  • Create double mutants combining mps2Δ with deletions of known stress response genes

  • Test synthetic phenotypes under various stress conditions

  • Examine potential compensatory mechanisms in the absence of MPS2

• Biochemical analyses:

  • Investigate post-translational modifications of MPS2 during stress (phosphorylation, etc.)

  • Identify stress-dependent protein-protein interactions using co-immunoprecipitation

  • Analyze potential changes in MPS2 protein levels or stability during stress responses

When designing these experiments, researchers should remember that A. gossypii differs from the model yeast S. cerevisiae in its persistent polarized growth pattern , which may influence how stress responses are integrated with growth and nuclear organization.

How can contradictory experimental results regarding MPS2 function be reconciled through methodological improvements?

When faced with contradictory experimental results regarding MPS2 function, researchers should implement these methodological strategies for reconciliation:

• Standardization of experimental conditions:

  • Establish consistent growth media, temperature, and culture age across experiments

  • Standardize protein expression and purification protocols

  • Define specific phenotypic metrics with quantitative parameters rather than qualitative descriptions

  • Account for strain background effects by using isogenic strains

• Comprehensive genetic analysis:

  • Create multiple independent knockout/knockdown strains using different selectable markers

  • Implement precise genome editing with CRISPR/Cas9 to avoid off-target effects

  • Use complementation tests with wild-type MPS2 to confirm phenotype specificity

  • Generate allelic series (partial loss-of-function) rather than relying solely on complete knockouts

• Multi-method validation:

  • Combine genetic, biochemical, and cell biological approaches to triangulate findings

  • Use multiple independent techniques to measure the same biological outcome

  • Implement in vitro reconstitution to complement in vivo observations

  • Verify protein-protein interactions through reciprocal co-immunoprecipitation, yeast two-hybrid, and in vitro binding assays

• Temporal and spatial resolution:

  • Employ time-lapse microscopy to capture dynamic processes rather than endpoint assays

  • Use inducible systems for precise temporal control of MPS2 expression or inactivation

  • Consider regional differences in hyphal development when analyzing phenotypes

  • Examine cell-to-cell variability within populations

• Computational modeling:

  • Develop mathematical models that integrate contradictory data into coherent frameworks

  • Use simulation approaches to identify parameter spaces where seemingly contradictory results can coexist

  • Implement sensitivity analyses to identify critical variables that might explain different experimental outcomes

By implementing these methodological improvements, researchers can build a more coherent understanding of MPS2 function in A. gossypii, potentially reconciling contradictory results by revealing context-dependent behaviors or previously unrecognized technical variables.

What emerging technologies could advance our understanding of MPS2 function in A. gossypii?

Several emerging technologies show promise for advancing our understanding of MPS2 function in A. gossypii:

• Super-resolution microscopy:

  • Techniques such as STORM, PALM, or STED microscopy could reveal nanoscale organization of MPS2 at spindle pole bodies and other subcellular structures

  • Multi-color super-resolution imaging could visualize MPS2 interactions with other proteins in situ

  • Live-cell super-resolution approaches could capture dynamic changes in MPS2 localization during hyphal growth and nuclear division

• CRISPR-based genomic tools:

  • CRISPRi for conditional knockdown of MPS2 with temporal precision

  • CRISPR activation systems to upregulate MPS2 expression

  • Base editing for introducing specific point mutations without double-strand breaks

  • Optogenetic CRISPR systems for spatiotemporal control of MPS2 expression

• Proximity labeling proteomics:

  • BioID or APEX2 fusion with MPS2 to identify proximally associated proteins in vivo

  • Comparative interactome analysis under different growth conditions or developmental stages

  • Identification of transient interactions that might be missed by conventional co-immunoprecipitation

• Single-cell transcriptomics and proteomics:

  • Analysis of cell-to-cell variability in mps2Δ mutants to identify compensatory mechanisms

  • Spatial transcriptomics to map gene expression changes along the hyphal length in relation to MPS2 function

  • Single-cell proteomics to quantify protein abundance and modification states

• Structural biology approaches:

  • Cryo-electron microscopy to determine the structure of MPS2 and its complexes

  • Integrative structural biology combining X-ray crystallography, NMR, and computational modeling

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes and protein interactions