Recombinant Ashbya gossypii Altered inheritance of mitochondria protein 37, mitochondrial (AIM37)

What is AIM37 and what is its biological significance?

AIM37 is a mitochondrial protein in Ashbya gossypii that belongs to a class of proteins identified through computational strategies specifically designed to uncover genes with altered inheritance of mitochondria. It plays a crucial role in mitochondrial structure and function, particularly in the formation and maintenance of mitochondrial contact sites . AIM37 was discovered alongside other AIM proteins during systematic genetic interaction mapping studies focused on mitochondrial function and inheritance patterns in filamentous fungi.

The biological significance of AIM37 extends beyond simple mitochondrial inheritance. This protein physically interacts with several other proteins including Fcj1, Aim5, and Aim13 to form a functional complex involved in maintaining mitochondrial architecture and functional integrity . The disruption of AIM37 can lead to alterations in mitochondrial morphology and function, which subsequently affects cellular metabolism, particularly in a filamentous fungus like A. gossypii that has high energy demands for growth and development.

How does AIM37 structurally and functionally differ from other AIM proteins?

AIM37 differs from other AIM proteins in several key aspects:

Structural Differences:

• Unlike AIM31, which has transmembrane characteristics , AIM37's structure is more consistent with a soluble protein that associates with membrane complexes

• While AIM43 contains specific domains like SPQTLEELARLKSLEDVDSSVIRKLINQRTEEVNAQNEAQ , AIM37 has a distinct amino acid sequence that enables its specific interactions with the MICOS complex

Functional Differences:

• AIM37 specifically interacts with the Fcj1 protein complex, which distinguishes it from AIM5 and AIM13 that have broader interaction profiles

• Unlike other AIM proteins that may have redundant functions, genetic studies suggest AIM37 has unique roles that cannot be compensated by other proteins in the mitochondrial network

Purification studies using tagged proteins have shown that when Aim37-FLAG is purified from mitochondrial extracts, it contains significant peptides/coverage of Fcj1, Aim5, and Aim13, indicating a tight physical interaction within this complex . This interaction pattern is distinct from other AIM proteins and suggests a specialized role in mitochondrial structure maintenance.

What methods are most effective for expressing recombinant AIM37 in laboratory settings?

Based on current research practices with A. gossypii proteins, the following methods are most effective for expressing recombinant AIM37:

Expression System Selection:

• E. coli expression systems are commonly used for recombinant AIM proteins, as demonstrated with AIM31

• For functional studies, A. gossypii itself can serve as an expression host using integrative cassettes rather than episomal vectors, which ensures stability in the multinucleated syncytium of A. gossypii

Vector Design Strategy:

• Implement a Golden Gate modular cloning system adapted for A. gossypii

• Design integrative cassettes containing:

  • Recombinogenic flanks targeting specific loci (e.g., ADR304W or AGL034C)

  • Selection marker (e.g., loxP-KanMX-loxP)

  • Strong promoter sequence (e.g., P GPD1)

  • AIM37 coding sequence

  • Efficient terminator sequence (e.g., PGK1)

Expression Optimization:

• Utilize the Dual Luciferase Reporter (DLR) Assay to optimize promoter selection for AIM37 expression

• Consider carbon source-regulatable promoters when temporal control of expression is desired

• For highest yields, grow cultures in rich media supplemented with appropriate carbon sources at 30°C

This approach has been validated for other proteins in A. gossypii and can be adapted specifically for AIM37 expression with appropriate modifications to account for protein-specific requirements.

What are the recommended purification protocols for maintaining AIM37 stability and activity?

For optimal purification of recombinant AIM37 while preserving its stability and activity, researchers should consider the following protocol:

Initial Cell Disruption:

• Harvest A. gossypii cells expressing AIM37 (preferably with a FLAG or His tag)

• Isolate crude mitochondria through differential centrifugation

• Solubilize mitochondrial membranes using digitonin (1-2%) which preserves protein-protein interactions better than harsher detergents

Affinity Purification:

• Apply the solubilized material to an appropriate affinity column:

  • For His-tagged AIM37: Nickel or cobalt affinity chromatography

  • For FLAG-tagged AIM37: Anti-FLAG antibody affinity chromatography

• Wash with increasing imidazole concentrations (for His-tag) or stringent washing buffers (for FLAG-tag)

• Elute with appropriate elution buffer (250-300 mM imidazole for His-tag or FLAG peptide for FLAG-tag)

Storage Recommendations:

• Store purified AIM37 in Tris-based buffer containing 50% glycerol

• Maintain at -20°C for short-term storage or -80°C for extended storage

• Avoid repeated freeze-thaw cycles; instead, prepare working aliquots stored at 4°C for up to one week

Quality Control:

• Verify purity by SDS-PAGE and immunoblotting

• Confirm identity through mass spectrometry analysis

• Assess activity through appropriate functional assays based on protein interaction studies

These protocols have been successfully applied to similar mitochondrial proteins from A. gossypii and should be effective for maintaining AIM37 stability and activity.

How does AIM37 interact with the MICOS complex and what is its significance?

AIM37 engages in critical interactions with the MICOS (Mitochondrial Contact Site and Cristae Organizing System) complex through both direct and indirect associations:

Interaction Mechanism:

• AIM37 physically interacts with Fcj1 (a core component of MICOS), as demonstrated by co-immunoprecipitation experiments where purifications of Aim37-FLAG contained significant peptides of Fcj1

• It forms a functional unit with Aim5 and Aim13, as all three proteins can be co-purified with each other

• These interactions are not merely structural but functionally significant as suggested by their genetic interaction profiles in the MITO-MAP

Functional Significance:

Research Implications:
When designing experiments to study AIM37-MICOS interactions, researchers should consider that disruption of these interactions leads to dramatic alterations in mitochondrial morphology including the formation of inner membrane septae and giant distorted mitochondria with irregular inner membranes . These phenotypes should be monitored using electron microscopy and fluorescence techniques to quantify the impact of experimental manipulations.

What experimental approaches reveal AIM37's role in mitochondrial inheritance and morphology?

To investigate AIM37's role in mitochondrial inheritance and morphology, researchers employ multiple complementary approaches:

Genetic Manipulation Strategies:

• Gene Deletion Studies:

  • Create Δaim37 knockout strains using homologous recombination

  • Compare mitochondrial inheritance patterns between wild-type and knockout strains

  • Quantify the frequency of mitochondrial inheritance defects

• Protein Tagging and Localization:

  • Express fluorescently-tagged AIM37 to track its subcellular localization

  • Monitor dynamic changes in AIM37 distribution during cell division

  • Correlate AIM37 positioning with mitochondrial segregation events

Morphological Analysis Methods:

• Electron Microscopy:

  • Thin-section TEM to visualize mitochondrial ultrastructure

  • Quantify cristae density, shape, and distribution

  • Measure the frequency of abnormal mitochondrial structures such as septae

• Confocal Microscopy with Mitochondrial Markers:

  • Use vital dyes (e.g., MitoTracker) or fluorescent proteins targeted to mitochondria

  • Perform time-lapse imaging to track mitochondrial dynamics

  • Quantify parameters such as mitochondrial network connectivity, organelle size, and distribution

Functional Assays:

• Respiration Measurements:

  • Oxygen consumption rates in intact cells and isolated mitochondria

  • Activity of individual respiratory complexes

  • Membrane potential measurements using fluorescent probes

• Mitochondrial Inheritance Quantification:

  • Count mitochondrial DNA nucleoids using DAPI staining

  • Track mitochondrial distribution during cell division

  • Calculate the efficiency of mitochondrial inheritance in daughter cells

Research has demonstrated that deletion of AIM37 leads to distinct functional deficiencies in mitochondria, including restrictions in growth on non-fermentable carbon sources, which is consistent with the reported alteration of inheritance of mitochondria . Additionally, combining AIM37 deletion with expression modifications of other MICOS components can lead to synthetic lethality on non-fermentable carbon sources, pointing to critical functional interactions .

How does AIM37 influence cardiolipin metabolism and mitochondrial membrane composition?

AIM37 exerts significant influence on cardiolipin metabolism and mitochondrial membrane composition through multiple mechanisms:

Impact on Cardiolipin Acyl Chain Composition:

• Research demonstrates that deletion of AIM37 in combination with alterations of MICOS components (particularly when His-tagged Mic12 or Mic26 are present) leads to significant changes in the acyl chain composition of cardiolipin

• Specifically, there is a shift toward longer and more saturated acyl chains, reminiscent of patterns observed in tafazzin-deficient cells

• These changes are remarkable as they mirror those described for mutants with deficiencies in tafazzin in various organisms, including those associated with Barth syndrome

Effect on Membrane Lipid Distribution:

Relationship with Respiratory Complexes:

• Cardiolipin is essential for the function of respiratory complexes

• The alteration in cardiolipin profiles correlates with the massive reduction of respiratory complexes observed in AIM37/MICOS mutants

• This establishes a mechanistic connection between AIM37, cardiolipin metabolism, and mitochondrial energetics

The following table summarizes the observed effects on mitochondrial components when AIM37 is deleted in combination with His-tagged MICOS subunits:

These findings suggest that AIM37 is part of a regulatory network that connects mitochondrial membrane architecture, lipid metabolism, and respiratory function .

What methodologies effectively assess AIM37's impact on respiratory chain function?

To comprehensively evaluate AIM37's impact on respiratory chain function, researchers should employ a multi-faceted methodological approach:

Biochemical Assays:

• Blue Native Gel Electrophoresis (BN-PAGE):

  • Isolate mitochondria from wild-type and AIM37-modified strains

  • Solubilize mitochondrial membranes with appropriate detergents (digitonin preferred)

  • Separate respiratory complexes and supercomplexes by BN-PAGE

  • Perform western blotting to identify specific complexes and quantify their abundance

• Spectrophotometric Enzyme Activity Assays:

  • Measure the activities of individual respiratory complexes (I-IV)

  • Assess ATP synthase (Complex V) function

  • Compare specific activities between wild-type and AIM37-modified strains

Physiological Measurements:

• Oxygen Consumption Analysis:

  • Use oxygen electrodes or plate-based respirometry (e.g., Seahorse XF Analyzer)

  • Measure basal, maximal, and reserve respiratory capacity

  • Determine the effect of specific inhibitors to assess individual complex contributions

• Membrane Potential Assessments:

  • Employ fluorescent probes (TMRM, JC-1) to measure mitochondrial membrane potential

  • Conduct flow cytometry or confocal microscopy for quantification

  • Perform time-lapse imaging to track dynamic changes in membrane potential

Growth and Viability Assays:

• Carbon Source Utilization:

  • Compare growth on fermentable (glucose) versus non-fermentable (lactate, glycerol) carbon sources

  • Quantify growth rates and maximum cell densities

  • Document morphological changes associated with different carbon sources

• Stress Response Evaluation:

  • Challenge cells with oxidative stress inducers (H₂O₂, paraquat)

  • Assess sensitivity to respiratory inhibitors (antimycin A, oligomycin)

  • Measure cell survival and recovery following stress exposure

Research has demonstrated that:

• AIM37 deletion in combination with His-tagged MICOS components (Mic12 or Mic26) leads to drastic reduction or complete absence of respiratory supercomplexes

• These mutants show strong reduction in steady-state levels of individual respiratory chain subunits

• There is an almost complete deficiency in subunit e (Su e) of the F₁F₀-ATP synthase

• These effects correlate with changes in cardiolipin composition, establishing a link between membrane composition and respiratory function

These methodologies collectively provide a comprehensive assessment of how AIM37 impacts respiratory chain function at multiple levels, from complex assembly to physiological performance.

How might researchers resolve contradictions in AIM37 functional studies?

When confronting contradictory findings in AIM37 functional studies, researchers should apply the following systematic approaches:

Standardization of Experimental Systems:

• Strain Background Considerations:

  • Verify the exact A. gossypii strain used (e.g., ATCC 10895, CBS 109.51, FGSC 9923, NRRL Y-1056)

  • Account for potential genetic differences between laboratory strains

  • Document any mutations that might have accumulated during strain maintenance

  • Consider that industrial strains optimized for riboflavin production may have altered mitochondrial function

• Growth Condition Harmonization:

  • Standardize media composition, particularly carbon sources

  • Control temperature, pH, and oxygen availability

  • Define growth phase for experiments (exponential vs. stationary)

  • Consider that A. gossypii can use various carbon sources including industrial byproducts

Methodological Refinement:

• Protein Expression and Tagging Strategy:

  • Compare N-terminal vs. C-terminal tags and their potential interference with function

  • Evaluate tag size impact (small epitope tags vs. larger fluorescent proteins)

  • Consider expression level effects (native promoter vs. overexpression)

  • Note that tag placement can affect protein localization or interaction

• Interaction Studies Approach:

  • Direct comparison between yeast two-hybrid, co-immunoprecipitation, and proximity labeling

  • Validation of interactions in both heterologous systems and native context

  • Consider that digitonin solubilization preserves interactions better than other detergents

Data Integration Framework:

• Multi-omics Data Integration:

  • Cross-reference transcriptomic, proteomic, and metabolomic datasets

  • Develop computational models to reconcile apparently contradictory findings

  • Consider that heterozygous mutations can have complex effects depending on nuclear state

• Phenotypic Hierarchy Analysis:

  • Establish primary vs. secondary effects through time-course studies

  • Use conditional expression systems to separate immediate from adaptive responses

  • Consider that multinucleated syncytium of A. gossypii may contain nuclei with different mutations

Researchers should be particularly attentive to the fact that A. gossypii is a multinucleate organism, and as demonstrated in the riboflavin-overproducing mutant analysis, it may contain multiple nuclei with different mutations . This can lead to heterogeneous populations of proteins and variable phenotypes depending on the proportion of nuclei carrying specific mutations.

What are the most promising directions for AIM37 research in relation to mitochondrial disease models?

The study of AIM37 has significant potential for advancing our understanding of mitochondrial diseases through several promising research directions:

Cardiolipin Metabolism and Barth Syndrome Models:

• Therapeutic Target Identification:

  • Explore whether modulation of AIM37 function can rescue cardiolipin abnormalities in tafazzin-deficient models

  • Investigate if AIM37 overexpression can compensate for defects in cardiolipin remodeling

  • Study synthetic interactions between AIM37 and tafazzin to identify potential compensatory mechanisms

• Membrane Architecture Regulation:

  • Determine how AIM37's interaction with the MICOS complex affects cristae formation

  • Investigate whether AIM37 can be targeted to stabilize mitochondrial membranes in disease states

  • Develop small molecules that modulate AIM37-MICOS interactions as potential therapeutic agents

Mitochondrial Dynamics and Quality Control:

• Fission/Fusion Machinery Interactions:

  • Map the functional relationship between AIM37 and mitochondrial dynamics proteins

  • Investigate AIM37's role in mitochondrial quality control mechanisms

  • Develop assays to measure how AIM37 affects mitochondrial turnover through mitophagy

• Metabolic Adaptation Mechanisms:

  • Explore AIM37's function in metabolic switching between respiratory and fermentative metabolism

  • Investigate how AIM37 contributes to mitochondrial adaptation during nutrient limitation

  • Determine whether AIM37's function is conserved in mammalian cells

Genetic Engineering Approaches:

• CRISPR/Cas9 Applications:

  • Develop precise gene editing strategies to introduce disease-relevant mutations in AIM37

  • Create cellular models with controlled expression of AIM37 variants

  • Establish high-throughput screening systems for compounds affecting AIM37 function

• Heterologous Expression Systems:

  • Optimize expression of human AIM37 homologs in A. gossypii

  • Develop dual expression systems for studying interaction partners

  • Create chimeric proteins to identify functional domains important for disease-relevant interactions

Potential Therapeutic Applications Table:

The observation that AIM37 deletion in combination with MICOS alterations mimics aspects of Barth syndrome pathology is particularly promising for developing new models of this disease . Furthermore, given A. gossypii's established role in industrial biotechnology , there's potential for developing this organism as a platform for screening therapeutic compounds targeting mitochondrial diseases.

What are the critical controls for studying AIM37 protein-protein interactions?

When investigating AIM37 protein-protein interactions, implementing comprehensive controls is essential for generating reliable and interpretable data:

Expression System Controls:

• Empty Vector Controls:

  • Include both untagged wild-type strain and empty vector transformants

  • Process these controls identically to experimental samples

  • Use for background subtraction in mass spectrometry analyses

• Tag-Only Controls:

  • Express the tag alone (e.g., FLAG, His) without AIM37

  • Identify non-specific interactions attributable to the tag

  • Compare elution profiles between tag-only and AIM37-tagged samples

Interaction Specificity Controls:

• Bait Stringency Series:

  • Perform purifications under increasing salt or detergent concentrations

  • Establish interaction strength hierarchy based on resistance to stringent conditions

  • Identify core versus peripheral interaction partners

• Reciprocal Tagging:

  • Tag multiple components of the putative complex (e.g., Fcj1, Aim5, Aim13)

  • Verify that AIM37 co-purifies with each tagged component

  • Confirm consistent stoichiometry across different bait proteins

Functional Validation Controls:

• Mutation Analysis:

  • Introduce point mutations in predicted interaction domains

  • Assess the impact on complex formation and function

  • Correlate biochemical interactions with functional outcomes

• Domain Mapping:

  • Create truncation mutants to identify minimal interaction domains

  • Express individual domains to test for direct binding

  • Use peptide competition assays to verify specific binding regions

Technical Method Controls:

• Sample Preparation Variables:

  • Compare different cell lysis methods (mechanical vs. enzymatic)

  • Test multiple detergents for membrane protein solubilization (digitonin, DDM, Triton X-100)

  • Assess the impact of protease inhibitor cocktail composition

• Crosslinking Validation:

  • If using crosslinking approaches, include non-crosslinked samples

  • Employ a range of crosslinker concentrations

  • Use both reversible and non-reversible crosslinkers

In published studies on A. gossypii mitochondrial proteins, researchers have effectively used FLAG-tagged proteins purified from digitonin-solubilized mitochondrial extracts, with untagged wild-type strains as controls . Mass spectrometry analysis included quantification of both the number of unique peptides and the percent coverage of detected proteins, providing robust metrics for interaction confidence .

How can researchers optimize recombinant AIM37 expression for structural studies?

Optimizing recombinant AIM37 expression for structural studies requires addressing several key challenges specific to mitochondrial membrane-associated proteins:

Expression Host Selection:

• Prokaryotic Systems:

  • E. coli BL21(DE3) or derivatives optimized for membrane proteins

  • Consider specialized strains with enhanced disulfide bond formation capabilities

  • Evaluate Lemo21(DE3) for tunable expression of potentially toxic proteins

  • This approach has been successful for other mitochondrial AIM proteins

• Eukaryotic Alternatives:

  • Pichia pastoris for proteins requiring post-translational modifications

  • Spodoptera frugiperda (Sf9) insect cells for complex eukaryotic proteins

  • Cell-free expression systems for difficult-to-express proteins

Vector and Construct Design:

• Fusion Partners for Solubility:

  • MBP (Maltose-Binding Protein) to enhance solubility

  • SUMO tag for improved folding and protease-mediated tag removal

  • Thioredoxin fusion for disulfide-rich proteins

• Expression Optimization Elements:

  • Codon optimization for the chosen expression host

  • Strong, inducible promoters with tunable expression levels

  • Inclusion of purification tags appropriate for structural studies (His6, FLAG, Twin-Strep)

Culture Condition Optimization:

• Temperature Modulation:

  • Reduced temperatures (16-25°C) to slow expression and improve folding

  • Heat shock protocols to induce chaperone expression before induction

  • Temporal regulation of induction intensity

• Media Formulation:

  • Enriched auto-induction media for controlled expression

  • Supplementation with specific lipids to stabilize membrane-associated domains

  • Addition of chemical chaperones (glycerol, arginine, trehalose)

Purification Strategy Refinement:

• Detergent Screening:

  • Test multiple detergents (DDM, LMNG, GDN, digitonin)

  • Implement detergent exchange during purification

  • Consider amphipols or nanodiscs for detergent-free final samples

• Chromatography Optimization:

  • Multi-step purification combining affinity, ion exchange, and size exclusion

  • On-column folding protocols for difficult proteins

  • Inclusion of stabilizing additives in all buffers

Stability Assessment Protocol:

For recombinant AIM37, researchers should pay particular attention to its association with membrane components and interaction partners. As observed with other AIM proteins, maintaining these interactions may be crucial for proper folding and stability . The storage recommendations used for commercial recombinant AIM proteins (Tris-based buffer with 50% glycerol, stored at -20°C or -80°C) provide a starting point, but structural studies will likely require more specialized buffer optimization.

How might AIM37 research contribute to understanding mitochondrial evolution?

AIM37 research offers several unique opportunities to advance our understanding of mitochondrial evolution through comparative genomics and functional analysis approaches:

Evolutionary Conservation Analysis:

• Phylogenetic Distribution:

  • AIM37 homologs can be traced across fungal lineages to identify conserved domains

  • Comparison between filamentous fungi like A. gossypii and unicellular yeasts like S. cerevisiae reveals evolutionary adaptations

  • This comparative approach is particularly valuable given that A. gossypii is evolutionarily close to unicellular yeasts but grows exclusively in a filamentous way

• Structural Domain Evolution:

  • Identification of functionally critical regions through multi-species sequence alignment

  • Correlation of sequence conservation with interaction interfaces

  • Mapping of lineage-specific insertions or deletions that may relate to specialized functions

Mitochondrial Architecture Evolution:

• Cristae Morphology Adaptation:

  • Compare AIM37's role in cristae formation across species with different mitochondrial morphologies

  • Investigate whether AIM37's function correlates with metabolic requirements in different organisms

  • A. gossypii represents an interesting model as it naturally overproduces riboflavin, which may place unique demands on mitochondrial function

• MICOS Complex Evolution:

  • Study how AIM37's integration with the MICOS complex varies across species

  • Identify lineage-specific interaction partners that may reflect adaptive specialization

  • Compare the phenotypic consequences of AIM37 deletion across evolutionary diverse fungi

Metabolic Adaptation Analysis:

• Respiratory vs. Fermentative Metabolism:

  • Investigate how AIM37's function relates to the balance between respiratory and fermentative metabolism

  • Explore whether AIM37 plays a role in the Crabtree effect (glucose repression of respiration)

  • This is particularly relevant as A. gossypii can effectively use various waste streams as carbon sources

• Riboflavin Production Correlation:

  • Examine potential links between AIM37 function and riboflavin biosynthesis

  • Study whether AIM37 influences FAD availability, which is derived from riboflavin

  • This connection is especially interesting given A. gossypii's industrial importance in riboflavin production

The compact organization of rRNA genes in A. gossypii (approximately 50 tandem repeat units of 8197 bp) compared to other fungi may provide insights into how mitochondrial function and inheritance mechanisms have co-evolved with nuclear genome organization. Additionally, the ability to engineer A. gossypii for enhanced production of compounds like FAD (derived from riboflavin) offers unique opportunities to study the evolutionary pressures on mitochondrial function in response to metabolic demands.

What are the technical challenges in studying AIM37 in different genetic backgrounds?

Investigating AIM37 across diverse genetic backgrounds presents several technical challenges that researchers must address through specialized methodological approaches:

Transformation and Gene Editing Challenges:

• Multinucleate Nature of A. gossypii:

  • The syncytial, multinucleate nature of A. gossypii complicates genetic manipulation

  • Transformation may initially yield heterokaryotic clones with both transformed and untransformed nuclei

  • Multiple rounds of spore isolation may be required to obtain homokaryotic clones

  • In some strains, particularly those with mutations affecting sporulation, obtaining homokaryotic transformants may be particularly challenging

• Integration Site Effects:

  • The genomic context of integration can significantly affect expression levels

  • Target integration to well-characterized neutral loci (e.g., ADR304W or AGL034C)

  • Confirm integration at the correct locus through analytical PCR and DNA sequencing

Expression Variability Management:

• Promoter Selection Considerations:

  • Different genetic backgrounds may respond differently to the same promoter

  • Test multiple promoters with different regulatory characteristics

  • Consider using the Dual Luciferase Reporter Assay to quantify promoter activity in each background

  • The 10 new promoters with different features, including carbon source-regulatable abilities, identified for A. gossypii provide valuable options

• Nuclear Heterogeneity Effects:

  • In strains with multiple nuclei containing different mutations, expression may be heterogeneous

  • The proportion of mutated reads in each gene can range from 21-75% as observed in riboflavin-overproducing mutants

  • Use single-cell approaches to account for nuclear heterogeneity

Phenotypic Analysis Complexities:

• Growth Media Requirements:

  • Different genetic backgrounds may have distinct nutritional requirements

  • Standardize media composition or develop strain-specific optimal media

  • Consider that some strains may be unable to utilize certain carbon sources efficiently

• Mitochondrial Phenotype Characterization:

  • Baseline mitochondrial morphology and function may vary between backgrounds

  • Establish strain-specific reference values for all mitochondrial parameters

  • Use internal controls within each strain rather than cross-strain comparisons

Data Integration Framework:

The development of effective marker recycling systems, such as Cre recombinase-mediated loxP-kanMX-loxP marker elimination , is particularly valuable for complex genetic manipulations in different backgrounds. These systems allow for sequential genetic modifications without accumulating multiple selection markers that might interfere with phenotypic analysis.

Additionally, researchers should consider that industrial strains of A. gossypii optimized for riboflavin production may contain multiple mutations affecting mitochondrial function , which could confound AIM37 functional studies if not properly accounted for.

What are the key bioinformatic resources for AIM37 analysis?

Researchers studying AIM37 can utilize the following bioinformatic resources for comprehensive analysis:

Sequence and Structure Analysis Tools:

• Protein Databases and Identifiers:

  • UniProt: The AIM37 entry can be accessed using accession number provided in product data sheets

  • NCBI Protein Database: Contains annotated AIM37 sequences with functional predictions

  • Saccharomyces Genome Database (SGD): While focused on yeast, contains valuable comparative information on AIM homologs

• Protein Structure Prediction:

  • AlphaFold2: Generate predicted structures for AIM37 and its interaction partners

  • SWISS-MODEL: Create homology models based on related proteins with known structures

  • I-TASSER: Integrated platform for structure and function prediction

Functional Analysis Resources:

• Mitochondrial Protein Databases:

  • MitoMiner: Integrated database of mitochondrial proteomics data

  • MitoCarta: Inventory of mammalian mitochondrial proteins with homology information

  • IMPI (Integrated Mitochondrial Protein Index): Comprehensive mitochondrial protein database

• Interaction Network Tools:

  • STRING: Protein-protein interaction networks with confidence scoring

  • BioGRID: Curated protein and genetic interactions

  • MICOS Complex databases: Specialized resources for cristae organizing system components

Genomic Analysis Platforms:

• Ashbya gossypii Resources:

  • AGD (Ashbya Genome Database): Comprehensive genome browser and analysis tools

  • Annotated genome sequence of reference strain ATCC 10895 / CBS 109.51 / FGSC 9923 / NRRL Y-1056

  • Transcriptomic datasets for A. gossypii under various conditions

• Comparative Genomics Tools:

  • Ensembl Fungi: Compare AIM37 across fungal species

  • OrthoMCL: Identify orthologous groups across species

  • FungiDB: Integrative genomic database for fungi

Experimental Design Resources:

• Gene Manipulation Databases:

  • CRISPR guide RNA design tools optimized for fungi

  • Codon optimization tools for A. gossypii expression

  • Promoter databases with characterized strength and regulation profiles

• Mitochondrial Methods Repositories:

  • Protocols for mitochondrial isolation from filamentous fungi

  • Optimized procedures for membrane protein solubilization

  • Imaging techniques for mitochondrial morphology in syncytial organisms

When analyzing AIM37 and its interactions, researchers should pay particular attention to the MITO-MAP genetic interaction data, which provides valuable insights into functional relationships between mitochondrial proteins . Additionally, the gene expression datasets from RNA-seq studies of A. gossypii can help identify co-regulated genes that may function in the same pathways as AIM37.

What standardized protocols ensure reproducibility in AIM37 functional studies?

To ensure reproducibility in AIM37 functional studies, researchers should adopt the following standardized protocols:

Strain Maintenance and Verification:

• Reference Strain Documentation:

  • Maintain precise records of strain provenance and history

  • Document the exact A. gossypii strain used (e.g., ATCC 10895, CBS 109.51)

  • Periodically verify strain identity through molecular markers

  • Create frozen stocks at early passages to prevent genetic drift

• Genome Verification Protocol:

  • Perform whole-genome sequencing or targeted sequencing of key loci

  • Compare to reference genome to identify any accumulated mutations

  • Document any deviations from reference sequence

  • Consider that industrial strains may contain multiple mutations

Gene Expression Standardization:

• Transcriptional Analysis Protocol:

  • RNA extraction optimized for filamentous fungi

  • qRT-PCR with validated reference genes for normalization

  • RNA-seq with appropriate depth for detecting low-abundance transcripts

  • Time-course analysis to capture dynamic expression patterns

• Protein Expression Quantification:

  • Standardized Western blotting protocol with validated antibodies

  • Inclusion of loading controls appropriate for mitochondrial proteins

  • Densitometry analysis using linear range of detection

  • Consider using SILAC or other quantitative proteomics approaches for higher precision

Functional Assays Standardization:

• Mitochondrial Isolation Protocol:

  • Standardized growth conditions before isolation

  • Gentle mechanical disruption methods for filamentous fungi

  • Density gradient purification to separate mitochondrial subpopulations

  • Quality control metrics for mitochondrial integrity

• Respiratory Function Analysis:

  • Oxygen consumption measurements under defined substrate conditions

  • Membrane potential assessment with calibrated fluorescent probes

  • Respiratory complex activity assays with specific substrates and inhibitors

  • Blue Native PAGE with consistent solubilization conditions

Microscopy Standards:

• Sample Preparation Protocol:

  • Standardized fixation methods for preserving mitochondrial morphology

  • Consistent immunolabeling procedures with validated antibodies

  • Mounting media selection to minimize artifacts

  • Use of fiducial markers for scale calibration

• Image Acquisition and Analysis:

  • Defined exposure settings and detector gain for quantitative imaging

  • Z-stack parameters for 3D reconstruction of mitochondrial networks

  • Blinded analysis to prevent observer bias

  • Automated image analysis workflows for morphological quantification

Data Reporting Requirements:

The adaptation of the Dual Luciferase Reporter (DLR) Assay for promoter analysis in A. gossypii using integrative cassettes provides an excellent model for standardized protocols. This approach demonstrated high efficiency in the analysis of promoter activity and can be adapted for studying AIM37 expression under various conditions.

Additionally, researchers should consider depositing their detailed protocols in repositories such as protocols.io to enhance reproducibility across different laboratories.