Recombinant Lodderomyces elongisporus Altered inheritance of mitochondria protein 11 (AIM11)

What is Lodderomyces elongisporus and how does it relate to AIM11 protein?

Lodderomyces elongisporus is a yeast species characterized by its distinctive elongated cell morphology, in contrast to the typical budding yeast morphology commonly observed in Candida species. At the genetic level, this fungus possesses a genome size of 15-16 Mb and belongs to the CTG clade, where the CUG codon translates as serine instead of leucine . Despite its classification within this clade, L. elongisporus exhibits notably lower virulence compared to other CTG clade members such as Candida albicans or Candida parapsilosis .

The AIM11 (Altered inheritance of mitochondria protein 11) is a protein encoded by the L. elongisporus genome. Based on its nomenclature and homology to similar proteins in other yeasts, AIM11 likely plays a role in mitochondrial inheritance or function during cell division. Understanding this protein's function may provide insights into the unique biological characteristics of L. elongisporus, particularly regarding its mitochondrial dynamics and potentially its elongated cellular morphology.

What are the basic storage and handling conditions for recombinant AIM11 protein?

For optimal maintenance of recombinant AIM11 protein activity, proper storage and handling are critical. The lyophilized powder should be stored at -20°C to -80°C upon receipt . When preparing for use, briefly centrifuge the vial to bring contents to the bottom before opening. For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

To prevent protein degradation during storage, aliquoting is necessary for multiple use. Adding glycerol to a final concentration of 5-50% before aliquoting is recommended for long-term storage . Once reconstituted, aliquots for immediate use can be stored at 4°C for up to one week, while the remaining protein should be stored at -20°C or -80°C .

How is recombinant AIM11 protein typically produced?

Recombinant Lodderomyces elongisporus AIM11 protein is typically produced using Escherichia coli as an expression system . The production process follows several key methodological steps:

• Gene Cloning: The AIM11 gene sequence is amplified from L. elongisporus genomic DNA or synthesized based on the reference sequence.

• Vector Construction: The gene is inserted into an expression vector that includes:

  • A strong promoter (typically T7 or similar)

  • An N-terminal His-tag sequence for purification

  • Appropriate antibiotic resistance markers

  • Origin of replication compatible with E. coli

• Transformation: The recombinant plasmid is transformed into an E. coli expression strain (commonly BL21(DE3) or derivatives).

• Expression Induction: Protein expression is induced, typically using IPTG for T7-based systems.

• Cell Lysis: Bacterial cells are harvested and lysed to release the recombinant protein.

• Affinity Purification: The His-tagged AIM11 protein is purified using immobilized metal affinity chromatography (IMAC).

• Quality Control: The purified protein undergoes SDS-PAGE analysis to confirm purity (>90%) and other quality control steps such as mass spectrometry to verify the correct sequence.

• Lyophilization: The purified protein is lyophilized for long-term stability and storage.

What is the significance of the His-tag in the recombinant AIM11 protein?

The His-tag in recombinant AIM11 protein serves multiple important research functions that facilitate its study:

• Purification Efficiency: The primary advantage of the His-tag is enabling efficient protein purification through immobilized metal affinity chromatography (IMAC). The polyhistidine sequence (typically 6-10 histidine residues) has high affinity for divalent metal ions like Ni²⁺ or Co²⁺ immobilized on chromatography resins. This allows for one-step purification with high specificity and yield .

• Detection Versatility: The His-tag provides a consistent epitope for detection using anti-His antibodies in various analytical techniques:

  • Western blotting

  • Enzyme-linked immunosorbent assay (ELISA)

  • Immunofluorescence microscopy

  • Flow cytometry

• Immobilization Capability: For interaction studies, His-tagged proteins can be immobilized on Ni-NTA surfaces for surface plasmon resonance (SPR) or other binding assays.

• Minimal Interference: The relatively small size of the His-tag (approximately 1 kDa) means it often causes minimal interference with protein structure and function compared to larger tags.

For structural or functional studies where the tag might potentially interfere, researchers should consider:

• Tag removal using specific proteases if cleavage sites were engineered between the tag and protein

• Control experiments comparing tagged versus untagged protein to assess any impact on function

• Placing the tag at either the N- or C-terminus based on structural predictions to minimize interference

What basic experimental methods can be used to study AIM11 protein function?

To elucidate AIM11 protein function, researchers can employ several foundational experimental approaches:

• Subcellular Localization Studies:

  • Fluorescence microscopy using GFP-tagged AIM11 or immunofluorescence with anti-His antibodies

  • Co-localization with mitochondrial markers (e.g., MitoTracker dyes)

  • Subcellular fractionation followed by Western blotting

• Protein-Protein Interaction Analyses:

  • Co-immunoprecipitation using His-tag pull-down

  • Yeast two-hybrid screening to identify interaction partners

  • Proximity labeling approaches (BioID or APEX)

  • Surface plasmon resonance for quantitative binding studies

• Genetic Manipulation Studies:

  • Generation of AIM11 knockout strains using CRISPR-Cas9

  • RNAi-mediated knockdown for partial loss-of-function

  • Overexpression studies to examine gain-of-function effects

  • Complementation experiments with mutated versions

• Functional Assays:

  • Mitochondrial distribution analysis during cell division

  • Mitochondrial membrane potential measurements

  • Oxygen consumption rate determination

  • ATP production assays

  • Mitochondrial morphology analysis

• Comparative Genomic Approaches:

  • Sequence analysis across fungal species to identify conserved domains

  • Phylogenetic analysis to trace evolutionary relationships

  • Comparison with characterized AIM proteins from other species

These methods provide a comprehensive foundation for investigating both the molecular and cellular functions of AIM11, particularly in relation to mitochondrial inheritance and potential roles specific to the elongated morphology of L. elongisporus .

What experimental designs are most appropriate for studying the role of AIM11 in mitochondrial inheritance?

For investigating AIM11's role in mitochondrial inheritance, researchers should implement sophisticated experimental designs that integrate multiple approaches:

• Comparative Genetic Analysis:

  • Generate isogenic strains differing only in AIM11 status (wild-type, knockout, point mutations)

  • Employ a factorial experimental design to test AIM11 function across different genetic backgrounds

  • Use quantitative trait locus (QTL) mapping to identify genetic modifiers of AIM11 function

• Live-Cell Imaging Strategies:

  • Implement time-lapse confocal microscopy with fluorescently labeled mitochondria and AIM11

  • Quantify mitochondrial inheritance patterns during cell division using automated image analysis

  • Apply photoactivatable fluorescent proteins to track specific mitochondrial subpopulations

• Molecular Dynamics Analysis:

  • Use fluorescence recovery after photobleaching (FRAP) to measure AIM11 mobility

  • Employ single-particle tracking to analyze AIM11 movement relative to mitochondria

  • Implement optogenetic approaches to manipulate AIM11 function with spatial and temporal precision

• Stepped-Wedge Experimental Design:

  • Adapt this design from implementation science to introduce AIM11 mutations sequentially across different yeast populations

  • This approach allows comparison of phenotypes before and after genetic manipulation while controlling for temporal effects

• Interrupted Time Series Analysis:

  • Track mitochondrial inheritance patterns over multiple generations with periodic AIM11 induction/repression

  • This design, borrowed from implementation science , enables detection of immediate versus delayed effects

• Structure-Function Analysis:

  • Create a library of AIM11 variants with domain deletions or point mutations

  • Perform complementation experiments in AIM11-knockout backgrounds

  • Correlate functional outcomes with structural features

• L. elongisporus-Specific Considerations:

  • Develop custom experimental protocols that account for the elongated cell morphology

  • Compare mitochondrial inheritance in L. elongisporus versus more conventional round yeasts

  • Examine potential relationships between AIM11 function and the species' non-utilization of L-Arabinose

These experimental designs should be implemented with appropriate controls, replication, and statistical analyses to ensure robust and reproducible findings about AIM11's role in mitochondrial inheritance.

How can we effectively analyze potential contradictions in data when studying AIM11 function?

When confronting contradictory data regarding AIM11 function, a systematic analytical approach is essential:

• Methodological Triangulation:

  • Examine AIM11 function using multiple independent techniques (genetic, biochemical, cell biological)

  • Determine if contradictions are technique-dependent or represent genuine biological complexity

  • Implement a mixed-methods approach combining quantitative and qualitative data

• Statistical Approaches for Contradictory Results:

  • Employ meta-analysis techniques to systematically compare results across experiments

  • Use Bayesian inference to update confidence in hypotheses as new data emerges

  • Implement sensitivity analyses to identify factors driving contradictory results

  • Apply appropriate statistical corrections for multiple comparisons

• Experimental Design Considerations:

  • Review experimental designs for potential confounding variables

  • Ensure appropriate randomization and blinding procedures

  • Consider implementation science frameworks for analyzing complex intervention effects

  • Evaluate whether contradictions appear consistently across different genetic backgrounds

• Causal Modeling Framework:

  • Develop structural causal models to represent hypothesized relationships

  • Generate testable predictions about AIM11 function under various scenarios

  • Use directed acyclic graphs (DAGs) to visualize potential confounding relationships

• Biological Context Analysis:

  • Consider whether contradictions reflect genuine condition-specific functions

  • Examine whether the unique CTG codon usage in L. elongisporus affects experimental outcomes

  • Investigate if the elongated morphology of L. elongisporus creates context-dependent AIM11 functions

• Collaborative Data Analysis:

  • Implement researcher triangulation by having multiple investigators analyze the same data

  • Consider preregistration of analysis plans to reduce confirmation bias

  • Use structured consensus methods when interpretations differ

• Computational Approaches:

  • Apply machine learning algorithms to identify patterns in complex datasets

  • Consider tabular foundation models for integrating diverse experimental results

  • Use simulation studies to test whether contradictions can be explained by known mechanisms

By systematically addressing contradictions rather than dismissing them, researchers can gain deeper insights into the complex biological roles of AIM11 and potentially discover novel aspects of mitochondrial inheritance mechanisms.

What are the methodological considerations for studying AIM11's interaction with other proteins?

When investigating AIM11's protein interactions, several sophisticated methodological considerations are critical for obtaining reliable and biologically meaningful results:

• Selection of Appropriate Detection Systems:

  • Proximity-Based Methods: BioID, APEX proximity labeling, or split-protein complementation assays

  • Direct Interaction Methods: Co-immunoprecipitation, pull-down assays, or FRET/BRET

  • High-Throughput Screening: Yeast two-hybrid or protein microarrays

  • Label-Free Detection: Surface plasmon resonance or isothermal titration calorimetry

• Tag Interference Mitigation:

  • Evaluate whether the N-terminal His-tag affects interactions by comparing with C-terminal tagged or untagged variants

  • Consider dual-tagging strategies for confirmatory experiments

  • Implement tag-removal systems using precision proteases when necessary

• Buffer and Environmental Conditions:

  • Optimize buffer composition to mimic mitochondrial microenvironment

  • Consider the influence of pH, ionic strength, and redox potential

  • Test interactions under different metabolic states relevant to mitochondrial function

• Expression System Considerations:

  • Account for the CTG codon translation difference in L. elongisporus when expressing in heterologous systems

  • Consider native expression levels to avoid artifacts from overexpression

  • Evaluate post-translational modifications that might occur in yeast but not in E. coli expression systems

• Validation Strategies:

  • Implement a multi-method validation approach for each interaction

  • Use reciprocal co-immunoprecipitation with differently tagged proteins

  • Confirm in vitro interactions with in vivo co-localization or functional studies

• Control Experiments:

  • Include non-binding mutants as negative controls

  • Use known interactors as positive controls

  • Implement scrambled or unrelated proteins of similar size and charge as specificity controls

  • Consider competition assays with untagged proteins to confirm specificity

• Quantitative Analysis:

  • Determine binding affinities when possible, not just binary interaction results

  • Assess stoichiometry of protein complexes

  • Evaluate kinetics of association and dissociation

• Context-Specific Considerations:

  • Examine interactions in the context of L. elongisporus's elongated morphology

  • Consider potential cell cycle-dependent interactions

  • Test whether interactions are affected by mitochondrial stress conditions

How can we design experiments to elucidate the role of AIM11 in the context of L. elongisporus's unique cellular morphology?

To investigate AIM11's function in relation to L. elongisporus's distinctive elongated morphology , experiments should intentionally bridge mitochondrial biology with cell shape regulation:

• Integrated Microscopy Approaches:

  • Implement multi-channel fluorescence microscopy with simultaneous visualization of:

    • AIM11 (fluorescently tagged)

    • Mitochondrial networks (MitoTracker or targeted fluorescent proteins)

    • Cell membrane/wall (membrane dyes)

    • Cytoskeletal elements (labeled actin and microtubules)

  • Apply quantitative image analysis to correlate AIM11 distribution with cell elongation axes

• Temporal Studies During Cell Division:

  • Employ time-lapse microscopy spanning complete cell cycles

  • Track mitochondrial inheritance patterns in relation to:

    • AIM11 localization dynamics

    • Changes in cell morphology during growth and division

    • Establishment of polarity axes in daughter cells

  • Quantify correlation coefficients between mitochondrial distribution and morphological parameters

• Comparative Genetic Approaches:

  • Generate AIM11 knockouts in both L. elongisporus and conventional round yeasts

  • Express L. elongisporus AIM11 in round yeasts to assess morphological effects

  • Create chimeric AIM11 proteins with domains from different species

  • Examine genetic interactions between AIM11 and known cell morphology regulators

• Cytoskeleton-Mitochondria Interaction Studies:

  • Examine the effect of cytoskeletal inhibitors on AIM11 localization

  • Perform co-immunoprecipitation of AIM11 with cytoskeletal proteins

  • Use subcellular fractionation to determine if AIM11 associates with both mitochondrial and cytoskeletal fractions

• Metabolic Context Integration:

  • Investigate whether the non-utilization of L-Arabinose by L. elongisporus has metabolic consequences that connect to:

    • AIM11 function

    • Mitochondrial activity

    • Cell morphology maintenance

  • Use the Arabinose (Loddy) test as a physiological marker in these studies

• Advanced Quantitative Analysis:

  • Apply machine learning algorithms to identify subtle correlations between:

    • Mitochondrial positioning

    • AIM11 localization patterns

    • Cell morphological parameters

  • Develop computational models predicting morphological outcomes based on AIM11 distribution

• Mechanical Force Studies:

  • Examine whether AIM11 plays a role in mitochondrial response to mechanical forces

  • Test if the elongated morphology creates unique mechanical environments for mitochondria

  • Use microfluidic devices to apply controlled mechanical forces to cells

These experimental approaches collectively address the potential specialized role of AIM11 in coordinating mitochondrial inheritance with the distinctive elongated morphology of L. elongisporus, potentially revealing novel aspects of organelle-cytoskeleton interactions in non-conventional yeast models.

What advanced imaging techniques would be most useful for studying AIM11 localization within cells?

For detailed examination of AIM11 localization, several cutting-edge imaging technologies offer complementary advantages:

• Super-Resolution Microscopy Techniques:

  • Structured Illumination Microscopy (SIM):

    • Provides ~100 nm resolution (2× improvement over conventional microscopy)

    • Maintains good compatibility with live-cell imaging

    • Ideal for tracking dynamic AIM11 movements in relation to mitochondrial networks

    • Allows for multi-color imaging to simultaneously visualize AIM11 and mitochondrial markers

  • Stimulated Emission Depletion (STED) Microscopy:

    • Achieves resolution down to ~30-70 nm

    • Enables detailed visualization of AIM11's distribution within mitochondrial subcompartments

    • Particularly valuable for examining potential clustering or domain formation

  • Single-Molecule Localization Microscopy (PALM/STORM):

    • Provides exceptional resolution (~20 nm)

    • Allows precise quantification of AIM11 molecule numbers and distribution

    • Capable of distinguishing between diffuse and clustered protein populations

    • Requires specialized fluorophores and fixation protocols

• Volumetric and Time-Resolved Imaging:

  • Lattice Light-Sheet Microscopy:

    • Enables long-term, high-speed volumetric imaging with minimal phototoxicity

    • Ideal for tracking AIM11 throughout complete cell division cycles

    • Provides excellent optical sectioning in the elongated L. elongisporus cells

    • Captures rapid dynamics that might be missed with slower imaging modalities

  • 4D Imaging (3D + time):

    • Tracks spatial reorganization of AIM11 and mitochondria during cellular processes

    • Essential for capturing the potentially complex mitochondrial inheritance patterns in elongated cells

• Correlative and Multimodal Approaches:

  • Correlative Light and Electron Microscopy (CLEM):

    • Combines fluorescence imaging of tagged AIM11 with ultrastructural context

    • Provides nanometer-resolution images of AIM11 localization relative to mitochondrial cristae

    • Particularly valuable for studying membrane contact sites

  • Expansion Microscopy:

    • Physically expands specimens to improve effective resolution

    • Compatible with conventional microscopes when super-resolution equipment is unavailable

    • Particularly useful for resolving AIM11 distribution in tight mitochondrial compartments

• Functional Imaging Approaches:

  • Fluorescence Lifetime Imaging Microscopy (FLIM):

    • Measures changes in AIM11's molecular environment through fluorescence lifetime changes

    • Can detect protein-protein interactions through FLIM-FRET

    • Less sensitive to concentration variations than intensity-based measurements

  • Fluorescence Correlation Spectroscopy (FCS):

    • Measures diffusion and binding dynamics of AIM11 in specific cellular locations

    • Provides quantitative data on molecular mobility and concentration

• Image Analysis Considerations:

  • Implement advanced computational image analysis:

    • Deconvolution algorithms to improve image quality

    • Machine learning approaches for automated detection of localization patterns

    • 3D reconstruction and rendering for volumetric visualization

    • Colocalization analysis with appropriate statistical validation

These advanced imaging approaches should be selected based on the specific research question, available equipment, and expertise, with consideration for the unique challenges presented by L. elongisporus's elongated morphology .

How might the CTG clade classification of L. elongisporus affect interpretation of AIM11 function?

The CTG clade classification of L. elongisporus, where the CUG codon translates as serine instead of leucine , has significant implications for AIM11 research and requires specialized methodological approaches:

• Expression System Considerations:

  • When expressing L. elongisporus AIM11 in standard laboratory organisms (e.g., S. cerevisiae or E. coli), CUG codons will be mistranslated

  • This could result in amino acid substitutions at critical functional sites, potentially affecting:

    • Protein folding and stability

    • Catalytic activity

    • Interaction capabilities

    • Subcellular localization signals

  • Researchers must use codon-optimized sequences that avoid CUG codons or employ CTG clade-specific expression systems

• Comparative Genomic Analysis Requirements:

  • Implement specialized sequence alignment algorithms that account for the alternative genetic code

  • Perform comparative analyses of AIM11 orthologs across:

    • CTG clade species (with same codon usage)

    • Conventional genetic code fungi

    • Other distant relatives

  • Identify conserved functional domains versus lineage-specific adaptations

  • Examine specifically whether CUG codons occur at functionally critical positions

• Evolutionary Context Interpretation:

  • Conduct phylogenetic analysis to trace how AIM11's sequence has evolved:

    • Before the CTG clade divergence

    • During the establishment of the alternative genetic code

    • After the divergence

  • This may reveal whether AIM11's role in mitochondrial inheritance has evolved differently within the CTG clade

• Experimental Design Adaptations:

  • Include parallel experiments in both CTG and standard genetic code systems

  • Create variant proteins with serine-to-leucine or leucine-to-serine substitutions at CUG positions

  • Assess functional differences between variants to isolate effects caused by the alternative genetic code

• Broader Evolutionary Questions:

  • Investigate potential links between altered genetic code evolution and mitochondrial inheritance mechanisms

  • Examine whether codons affecting AIM11 may have changed during L. elongisporus divergence

  • Consider whether the alternative genetic code creates selective pressure on mitochondrial function

• Methodological Validation Approaches:

  • Confirm protein sequences by mass spectrometry to verify actual amino acid incorporation

  • Implement ribosome profiling to examine translation efficiency at CUG codons

  • Consider native purification from L. elongisporus for critical experiments

• Computational Prediction Adjustments:

  • Ensure that protein structure prediction algorithms account for the alternative genetic code

  • Adjust homology modeling parameters to reflect accurate amino acid substitutions

  • Re-evaluate functional domain predictions considering the proper translation

The CTG clade classification represents both a challenge and opportunity in AIM11 research. While it complicates heterologous expression and functional comparisons, it also provides a valuable system for studying how genetic code alterations influence protein evolution and function, particularly in proteins involved in fundamental cellular processes like mitochondrial inheritance.