Recombinant Ajellomyces capsulata Formation of crista junctions protein 1 (FCJ1)

What is FCJ1 protein and what is its primary function in cellular biology?

FCJ1 (Formation of crista junctions protein 1), also known as Mitofilin, is a protein predominantly located at crista junctions in mitochondria. It plays a crucial role in forming and maintaining the architecture of these junctions, which are tubular invaginations of the inner mitochondrial membrane that connect the inner boundary with the cristae membrane . These architectural elements are critical for proper mitochondrial function and energy production. In Ajellomyces capsulata (the teleomorphic or sexual stage of Histoplasma capsulatum), FCJ1 exhibits structural and functional similarities to mitofilin proteins found in other eukaryotes, including mammals . The protein is essential for maintaining mitochondrial ultrastructure and, consequently, for cellular energy metabolism and homeostasis.

What is the relationship between Ajellomyces capsulata and Histoplasma capsulatum in the context of FCJ1 research?

Ajellomyces capsulata represents the teleomorphic (sexual) stage of the dimorphic fungal pathogen Histoplasma capsulatum. While Histoplasma capsulatum refers to the asexual (anamorphic) stage, Ajellomyces capsulata specifically denotes the sexual reproductive form resulting from the mating of (+) and (-) types . This distinction is important in research contexts because the sexual stage may express certain proteins, including FCJ1 variants, differently than the asexual form. The sexual reproduction in this species is regulated by a specialized genomic region known as the mating-type (MAT1) locus, with isolates containing either MAT1-1 or MAT1-2 idiomorphs encoding different transcription factors . Understanding this relationship provides context for studying FCJ1 across different life stages of the organism and may inform investigations into how mitochondrial structure varies during the fungal life cycle.

What are the key structural domains of FCJ1 and how do they relate to its function?

FCJ1 protein contains several distinct structural domains, with the C-terminal domain being the most evolutionarily conserved and functionally significant. The protein structure includes:

• An N-terminal mitochondrial targeting sequence

• A transmembrane domain anchoring the protein to the inner mitochondrial membrane

• A coiled-coil domain involved in protein-protein interactions

• The highly conserved C-terminal domain

The C-terminal domain is essential for FCJ1 function and proper formation of crista junctions. In experimental studies, absence of this domain results in strongly impaired and irregular CJ formation with stacked cristae developing as a consequence . This domain mediates interactions with full-length FCJ1, suggesting a role in oligomer formation that stabilizes the protein complex at crista junctions. Additionally, the C-terminal domain interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB/SAM) complex, indicating its importance in establishing contact sites between the inner and outer mitochondrial membranes .

What are the optimal conditions for reconstituting recombinant FCJ1 protein for experimental use?

For optimal reconstitution of lyophilized recombinant FCJ1 protein from Ajellomyces capsulata:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% for long-term storage stability

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

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep at -20°C or -80°C

The protein stability is optimal in Tris-based buffer systems with pH 8.0 . When designing experiments, consider that the recombinant protein typically contains a His-tag at the N-terminus, which may need to be accounted for in certain interaction studies or assays where tag interference might be a concern. The purity of commercially available recombinant FCJ1 is typically greater than 90% as determined by SDS-PAGE, making it suitable for most research applications .

How can researchers verify the functional integrity of recombinant FCJ1 in experimental systems?

To verify the functional integrity of recombinant FCJ1, researchers should employ multiple complementary approaches:

• Structural integrity assessment:

  • SDS-PAGE analysis to confirm molecular weight and purity

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Limited proteolysis to assess proper folding

• Interaction verification:

  • Co-immunoprecipitation experiments to detect interaction with known binding partners (particularly Tob55 of the TOB/SAM complex)

  • Pull-down assays to confirm oligomerization capability through interaction with full-length FCJ1

  • FRET or other proximity assays to verify spatial relationships with partner proteins

• Functional complementation:

  • Expression of recombinant FCJ1 in FCJ1-deficient cell models

  • Electron microscopy to evaluate rescue of mitochondrial crista junction morphology

  • Measurement of mitochondrial membrane potential and respiratory capacity

The key functional readout is the ability of the recombinant protein to interact with the TOB/SAM complex and stabilize crista junctions in proximity to the outer membrane . Researchers should be particularly attentive to the integrity of the C-terminal domain, as this region is critical for proper protein function in maintaining mitochondrial ultrastructure.

What methods are recommended for studying FCJ1 interactions with the TOB/SAM complex?

For investigating the critical interactions between FCJ1 and the TOB/SAM complex, researchers should consider the following methodological approaches:

• In vitro binding assays:

  • GST pull-down or His-tag pull-down assays using purified recombinant FCJ1 and TOB/SAM components

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity constants

  • Isothermal titration calorimetry (ITC) to quantify thermodynamic parameters of the interaction

• Structural studies:

  • Chemical cross-linking followed by mass spectrometry to identify specific interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map binding regions

  • Cryo-electron microscopy of the FCJ1-TOB/SAM complex to visualize the interaction architecture

• Cellular localization and interaction:

  • Proximity ligation assays to visualize interactions in situ

  • FRET or BRET assays to monitor interactions in living cells

  • Immunofluorescence microscopy with super-resolution techniques to visualize co-localization

• Functional consequences:

  • Site-directed mutagenesis of the C-terminal domain to identify critical residues for TOB/SAM interaction

  • Assessment of mitochondrial morphology following disruption of specific interaction sites

  • Evaluation of β-barrel protein assembly in the context of FCJ1-TOB/SAM interaction perturbations

These methods should be interpreted with the understanding that the association of the TOB/SAM complex with contact sites depends on the presence of FCJ1, and that while the biogenesis of β-barrel proteins is not significantly affected in the absence of FCJ1, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in crista junctions .

What are the technical challenges in studying FCJ1's role in mitochondrial membrane architecture using recombinant protein?

Studying FCJ1's role in mitochondrial membrane architecture using recombinant protein presents several significant technical challenges:

• Membrane protein reconstitution:

  • FCJ1 is an inner mitochondrial membrane protein, making it difficult to study in isolation

  • Requires artificial membrane systems or liposomes to recapitulate the native environment

  • Detergent selection for solubilization can significantly impact protein folding and function

• Complex multi-protein interactions:

  • FCJ1 functions within a network of protein complexes at crista junctions

  • Reconstituting these complex interaction networks in vitro is technically demanding

  • The interactions with the TOB/SAM complex and other mitochondrial proteins may require specific lipid compositions and membrane curvatures

• Structural constraints:

  • The protein contains multiple domains with different properties, including transmembrane regions

  • Maintaining proper folding during recombinant expression and purification is challenging

  • The C-terminal domain's interaction with full-length FCJ1 suggests oligomerization is important for function

• Functional assays:

  • Difficulty in designing quantitative assays for crista junction formation in vitro

  • Requires sophisticated imaging techniques like electron tomography to visualize membrane architecture

  • Correlating in vitro findings with in vivo mitochondrial ultrastructure remains challenging

• Species-specific considerations:

  • The fungal FCJ1 may have structural or functional differences compared to mammalian homologs

  • Recombinant fungal proteins may not correctly interact with mammalian experimental systems

  • Differences in post-translational modifications between expression systems and native context

To address these challenges, researchers often need to employ complementary approaches combining in vitro reconstitution systems with cellular models and advanced imaging techniques. Cryo-electron tomography of mitochondria from cells expressing recombinant FCJ1 variants can provide valuable insights into the protein's role in membrane architecture that purely biochemical approaches might miss.

How does FCJ1 oligomerization contribute to crista junction stability, and what methodologies can best characterize these oligomeric states?

FCJ1 oligomerization is critically important for stabilizing crista junctions, with the C-terminal domain playing a key role in mediating these interactions. To fully characterize the nature and functional significance of these oligomeric states, researchers should employ the following methodologies:

• Analytical ultracentrifugation and size-exclusion chromatography:

  • Determine the stoichiometry of FCJ1 oligomers under varying conditions

  • Assess the impact of the C-terminal domain on oligomerization by comparing full-length and truncated variants

  • Evaluate the stability of oligomeric complexes in different buffer compositions

• Cross-linking mass spectrometry:

  • Identify specific residues involved in the oligomerization interface

  • Map interaction networks within larger FCJ1-containing complexes

  • Distinguish between direct and indirect interactions in multi-protein assemblies

• Single-molecule techniques:

  • Fluorescence correlation spectroscopy to determine oligomer size distributions

  • Single-molecule FRET to measure conformational changes during oligomerization

  • Super-resolution microscopy to visualize oligomer distribution in mitochondrial membranes

• Functional correlation studies:

  • Site-directed mutagenesis of potential oligomerization interfaces

  • Correlation between oligomerization propensity and crista junction formation

  • Assessment of how oligomerization affects interactions with the TOB/SAM complex

The model emerging from current research suggests that FCJ1 forms oligomeric complexes through C-terminal domain interactions, creating a scaffold that stabilizes crista junctions by anchoring them to contact sites near the outer membrane through interaction with the TOB/SAM complex . The oligomeric nature of FCJ1 likely creates a ring-like structure that defines and maintains the tubular architecture of crista junctions, with disruption of oligomerization leading to altered mitochondrial ultrastructure.

What is the significance of FCJ1 in Histoplasma capsulatum pathogenesis and host-pathogen interactions?

The role of FCJ1 in Histoplasma capsulatum pathogenesis represents an emerging area of research with implications for understanding fungal virulence mechanisms. While direct evidence linking FCJ1 to pathogenicity is still developing, several lines of research suggest potential significance:

• Mitochondrial dynamics during morphological switching:

  • Histoplasma capsulatum undergoes dimorphic switching between yeast and mycelial forms during infection

  • Mitochondrial remodeling accompanies this transition, suggesting FCJ1 involvement in adaptations to host environment

  • Proper mitochondrial function supported by FCJ1 may be critical for survival within macrophages

• Energy metabolism during infection:

  • FCJ1's role in maintaining cristae architecture affects respiratory capacity

  • During intracellular parasitism, the fungus must adapt to nutrient-limited conditions

  • Efficient mitochondrial function may represent a virulence determinant for persistent infection

• Potential as a therapeutic target:

  • The structural differences between fungal FCJ1 and mammalian mitofilin might be exploited

  • Compounds disrupting FCJ1 function could potentially inhibit fungal growth with minimal host toxicity

  • The conserved C-terminal domain represents a potential target for antifungal development

• Host immune recognition:

  • Mitochondrial proteins can be exposed during fungal cell stress or death

  • FCJ1 could potentially serve as a pathogen-associated molecular pattern (PAMP)

  • Antibodies against FCJ1 might be detectable in patients with histoplasmosis

When studying FCJ1 in the context of Histoplasma/Ajellomyces pathogenesis, researchers should consider both the sexual (Ajellomyces) and asexual (Histoplasma) stages of the fungus, as the teleomorphic stage may express FCJ1 variants that could influence mitochondrial function in environmentally adapted forms of the pathogen .

How can researchers design experiments to investigate the effects of FCJ1 dysfunction on mitochondrial respiration and cellular metabolism?

Designing robust experiments to investigate FCJ1 dysfunction effects on mitochondrial respiration and cellular metabolism requires a multi-parametric approach:

• Genetic manipulation systems:

  • CRISPR/Cas9-mediated FCJ1 knockout or knockdown

  • Site-directed mutagenesis targeting the C-terminal domain

  • Conditional expression systems to study acute vs. chronic FCJ1 depletion

  • Complementation with wild-type or mutant FCJ1 variants

• High-resolution respirometry:

  • Measure oxygen consumption rates in intact cells and isolated mitochondria

  • Assess respiratory complex activities individually and in combination

  • Determine respiratory control ratios as indicators of coupling efficiency

  • Evaluate capacity for OXPHOS vs. glycolytic ATP production

• Mitochondrial membrane potential and ROS production:

  • Fluorescent probes (TMRM, JC-1) for membrane potential measurement

  • Live-cell imaging to track dynamic changes in potential

  • Quantification of ROS production using targeted sensors

  • Correlation of membrane potential stability with FCJ1 levels/function

• Metabolomic analysis:

  • Targeted metabolomics focused on TCA cycle intermediates

  • Stable isotope labeling to track metabolic flux

  • Measurement of NAD+/NADH and ATP/ADP ratios

  • Assessment of amino acid and fatty acid metabolism

• Structural-functional correlations:

  • Electron microscopy to quantify crista density and morphology

  • Super-resolution microscopy to visualize respiratory complex organization

  • Correlation of ultrastructural features with respiratory parameters

  • Assessment of mitochondrial network dynamics and fragmentation

Experimental design should include appropriate controls, such as comparison with TOB/SAM complex downregulation, which has been shown to lead to altered cristae morphology and moderate reduction in crista junctions . Data interpretation should acknowledge that FCJ1 dysfunction may have both direct effects on respiratory efficiency through altered cristae architecture and indirect effects through disrupted protein import pathways.

What methodological approaches can be used to study the relationship between FCJ1 and the maintenance of mitochondrial DNA integrity?

The relationship between FCJ1 and mitochondrial DNA (mtDNA) integrity represents an important but under-explored aspect of mitochondrial biology. Researchers investigating this connection should consider these methodological approaches:

• Quantification of mtDNA copy number and integrity:

  • qPCR-based methods to measure mtDNA:nuclear DNA ratios

  • Long-range PCR to detect large-scale deletions

  • Next-generation sequencing to identify point mutations and heteroplasmy

  • Single-molecule real-time sequencing for detailed mutation analysis

• Nucleoid organization and dynamics:

  • Super-resolution microscopy to visualize mtDNA nucleoid structure

  • Immunofluorescence co-localization of FCJ1 with nucleoid proteins (TFAM, Twinkle)

  • Live-cell imaging to track nucleoid mobility in relation to crista structures

  • ChIP-seq approaches to map protein-mtDNA interactions

• mtDNA replication and transcription:

  • BrdU incorporation assays to measure mtDNA synthesis rates

  • RNA-seq for mitochondrial transcriptome analysis

  • Run-on assays to measure transcription initiation and elongation

  • Analysis of mitochondrial ribosome loading and translation efficiency

• Oxidative damage and repair pathways:

  • 8-oxo-dG immunostaining to detect oxidative DNA damage

  • Activity assays for mitochondrial DNA repair enzymes

  • Protein-protein interaction studies between FCJ1 and DNA maintenance factors

  • Assessment of mitochondrial unfolded protein response activation

• Functional genomic screening:

  • Synthetic genetic interaction screens with FCJ1 and mtDNA maintenance genes

  • Suppressor screens to identify compensatory pathways

  • Chemical-genetic screens to find compounds that specifically affect FCJ1-deficient cells

  • Targeted CRISPR library screening focused on mitochondrial functions

The working hypothesis in these studies would be that FCJ1-dependent crista architecture influences nucleoid positioning and protection, potentially affecting mtDNA exposure to reactive oxygen species generated by the respiratory chain. Comparative analysis between wild-type and FCJ1-deficient cells under various metabolic and stress conditions would help elucidate how crista junction integrity contributes to mtDNA stability and inheritance.

What emerging technologies might advance our understanding of FCJ1 structure-function relationships in mitochondrial membrane dynamics?

Several cutting-edge technologies hold promise for deepening our understanding of FCJ1's role in mitochondrial membrane dynamics:

• Cryo-electron tomography with focused ion beam milling:

  • Enables visualization of FCJ1 in its native membrane environment at near-atomic resolution

  • Allows 3D reconstruction of crista junction architecture in wild-type and mutant cells

  • Can reveal the spatial organization of FCJ1 complexes in relation to other mitochondrial structures

  • Provides insights into how FCJ1 oligomerization shapes membrane curvature

• In-cell structural biology:

  • Proximity-dependent labeling (BioID, APEX) to map FCJ1's interaction network in living cells

  • In-cell NMR to probe structural dynamics under physiological conditions

  • Integrative structural modeling combining multiple data sources

  • Single-particle tracking to measure FCJ1 mobility within membranes

• Artificial organelle systems:

  • Reconstitution of minimal mitochondrial membranes with purified components

  • Bottom-up assembly of crista-like structures with defined protein composition

  • Microfluidic systems to manipulate membrane curvature and tension

  • Direct visualization of FCJ1-induced membrane remodeling in real-time

• Advanced genetic tools:

  • Optogenetic control of FCJ1 oligomerization or localization

  • Split protein complementation to visualize FCJ1 interactions dynamically

  • Base editing for precise modification of FCJ1 at endogenous loci

  • Tissue-specific or inducible manipulation of FCJ1 in model organisms

• Computational approaches:

  • Molecular dynamics simulations of FCJ1 in membrane environments

  • Machine learning analysis of crista junction morphological features

  • Systems biology modeling of how FCJ1 perturbations propagate through mitochondrial networks

  • Evolutionary analysis to identify co-evolving residues important for function

These technologies would help address fundamental questions about how FCJ1 oligomers assemble to stabilize crista junctions, how these structures respond to changes in mitochondrial energetics, and how the C-terminal domain interactions with the TOB/SAM complex contribute to the spatial organization of the mitochondrial membranes .

What potential therapeutic applications might arise from a deeper understanding of FCJ1 function in mitochondrial dynamics?

Research on FCJ1 and its role in mitochondrial architecture has potential therapeutic implications in several areas:

• Fungal infectious disease treatment:

  • Development of selective inhibitors targeting fungal FCJ1 but not mammalian mitofilin

  • Identification of compounds disrupting FCJ1-specific protein interactions

  • Creation of combination therapies targeting mitochondrial function in pathogenic fungi

  • Exploitation of differences in FCJ1 structure between Histoplasma and human cells

• Mitochondrial dysfunction disorders:

  • Gene therapy approaches to correct FCJ1 mutations or expression levels

  • Small molecules stabilizing beneficial FCJ1 interactions

  • Peptide-based therapeutics mimicking functional domains of FCJ1

  • Metabolic interventions to compensate for altered cristae architecture

• Neurodegenerative diseases:

  • Modulation of FCJ1 function to maintain mitochondrial integrity in neurons

  • Protection against mitochondrial fragmentation in Alzheimer's or Parkinson's disease

  • Enhancement of mitochondrial quality control through FCJ1-dependent pathways

  • Biomarkers based on FCJ1 integrity or modifications in disease states

• Cancer metabolism:

  • Targeting cancer-specific alterations in cristae architecture

  • Combination with existing therapies affecting mitochondrial function

  • Exploitation of metabolic vulnerabilities in tumors with altered FCJ1 function

  • Development of imaging agents to visualize mitochondrial structural changes in vivo

• Aging and senescence:

  • Interventions to maintain optimal crista junction structure during aging

  • Protection of mtDNA integrity through FCJ1-dependent mechanisms

  • Modulation of mitochondrial dynamics to promote healthy aging

  • Biomarkers of mitochondrial structural integrity in aging populations

The therapeutic development process would benefit from comparative studies of FCJ1 across species, detailed characterization of the C-terminal domain interactions, and development of high-throughput screening assays to identify compounds affecting FCJ1 function or crista junction formation. The unique relationship between FCJ1 and the TOB/SAM complex suggests potential for targeting this interaction in specific disease contexts .

How might integrative multi-omics approaches advance our understanding of FCJ1's role in cellular stress responses and adaptation?

Integrative multi-omics approaches offer powerful frameworks for understanding FCJ1's comprehensive role in cellular stress responses and adaptation:

• Multi-level omics integration:

  • Genomics: Identification of genetic variants affecting FCJ1 function across populations

  • Transcriptomics: Analysis of gene expression changes in FCJ1-deficient cells under stress

  • Proteomics: Quantification of protein abundance, post-translational modifications, and interaction networks

  • Metabolomics: Profiling of metabolic signatures associated with FCJ1 dysfunction

  • Lipidomics: Characterization of membrane lipid composition changes affecting crista architecture

• Temporal dynamics analysis:

  • Time-course experiments capturing acute vs. chronic adaptation to FCJ1 perturbation

  • Pulse-chase proteomics to measure protein turnover rates

  • Metabolic flux analysis using stable isotope labeling

  • Single-cell trajectories during stress responses and recovery phases

• Spatial organization insights:

  • Spatial proteomics to map protein redistribution within mitochondrial compartments

  • Sub-organellar metabolite profiling to identify localized metabolic changes

  • Correlation of structural changes with local alterations in protein composition

  • Visualization of signaling pathway activation in relation to mitochondrial networks

• Systems-level network analysis:

  • Construction of integrated networks connecting FCJ1 to mitochondrial and cellular functions

  • Identification of hub proteins and key regulatory nodes affected by FCJ1 status

  • Comparative network analysis across different stress conditions

  • Predictive modeling of cellular responses to combined stressors

• Translational applications:

  • Identification of biomarkers for mitochondrial stress responses

  • Discovery of targetable nodes for therapeutic intervention

  • Development of companion diagnostics for mitochondrial-targeted therapies

  • Personalized approaches based on individual variation in stress response networks

The integration of these diverse data types would provide a comprehensive view of how FCJ1-dependent mitochondrial architecture influences cellular adaptation to stress, potentially revealing unexpected connections between crista junction integrity and broader cellular processes such as inflammatory responses, cell death regulation, and metabolic reprogramming under challenging conditions.

What are the current consensus views and remaining controversies regarding FCJ1's role in mitochondrial function?

The current scientific consensus regarding FCJ1's role in mitochondrial function encompasses several well-established principles, while important questions remain unresolved:

Consensus views:

• FCJ1 is essential for the formation and maintenance of crista junctions in mitochondria, with its absence leading to severely altered cristae morphology .

• The C-terminal domain of FCJ1 is the most conserved region across species and is critical for protein function, mediating interactions with both full-length FCJ1 and components of the TOB/SAM complex .

• FCJ1 functions in stabilizing crista junctions in close proximity to the outer membrane, creating organized contact sites important for mitochondrial architecture .

• The protein plays an important role in maintaining mitochondrial function, with its absence affecting respiratory efficiency and potentially other mitochondrial processes.

Remaining controversies and open questions:

• The precise molecular mechanism by which FCJ1 induces membrane curvature at crista junctions remains debated.

• The stoichiometry and complete composition of FCJ1-containing complexes at crista junctions are not fully characterized.

• The extent to which FCJ1 functions are conserved between fungal species (like Ajellomyces capsulata) and mammals is unclear.

• The regulatory mechanisms controlling FCJ1 expression, localization, and activity during different cellular states are poorly understood.

• The potential roles of FCJ1 in processes beyond maintaining cristae architecture, such as mtDNA maintenance, apoptosis regulation, or response to specific stressors, remain to be fully elucidated.

These areas of consensus and controversy define the current research landscape for FCJ1, with ongoing work focused on resolving the structural biology of FCJ1 complexes, the dynamic regulation of crista junctions, and the integration of these structures with broader mitochondrial and cellular functions.

What standardized methods should researchers adopt when comparing results across different experimental systems studying FCJ1?

To ensure comparability and reproducibility in FCJ1 research across different experimental systems, researchers should adopt these standardized methods:

• Protein characterization standards:

  • Consistent nomenclature distinguishing full-length FCJ1 from domain-specific constructs

  • Standardized purity assessment methods (minimum 90% by SDS-PAGE)

  • Defined buffer compositions for storage and experimental use

  • Validation of proper folding using circular dichroism or other structural techniques

• Expression system documentation:

  • Detailed reporting of expression vectors, tags, and their positions

  • Characterization of post-translational modifications present or absent

  • Assessment of oligomeric state in the recombinant preparation

  • Verification of functional activity through standardized assays

• Mitochondrial morphology quantification:

  • Standardized electron microscopy sample preparation protocols

  • Objective quantification methods for crista density, length, and junction diameter

  • Statistical approaches for analyzing morphological variation

  • Minimum reporting requirements for sample sizes and representative images

• Functional assays:

  • Standardized protocols for measuring respiratory capacity

  • Defined methods for assessing membrane potential with specific probes

  • Consistent approaches to measuring interaction with TOB/SAM complex components

  • Reproducible assays for evaluating oligomerization and membrane association

• Genetic manipulation reporting:

  • Complete description of knockout/knockdown strategies and verification

  • Quantification of residual FCJ1 levels in partial depletion models

  • Documentation of potential compensatory responses

  • Detailed complementation protocols for rescue experiments

By adopting these standardized approaches, researchers can more effectively compare results across different model systems, from yeast to fungi to mammalian cells, enhancing the translational value of findings and accelerating progress in understanding FCJ1's roles in health and disease. This standardization is particularly important when working with recombinant proteins like those described in commercial preparations of Ajellomyces capsulata FCJ1 .

How might researchers effectively bridge the gap between structural studies of FCJ1 and its functional significance in cellular and organismal physiology?

Bridging the gap between structural insights into FCJ1 and its broader functional significance requires integrative approaches that connect molecular mechanisms to physiological outcomes:

• Structure-guided functional analysis:

  • Generation of precise point mutations based on structural data

  • Domain-swap experiments between species to identify conserved functional elements

  • Structure-based design of interaction disruptors or enhancers

  • Correlation of structural features with evolutionary conservation patterns

• Multi-scale imaging approaches:

  • Correlated light and electron microscopy to link protein localization to ultrastructure

  • In situ structural biology techniques like cryo-electron tomography

  • Live-cell super-resolution imaging to track dynamic structural changes

  • Whole-organism imaging to visualize mitochondrial network changes in tissues

• Physiological readouts at multiple levels:

  • Cell-based assays measuring bioenergetic parameters

  • Tissue-specific phenotyping in model organisms with FCJ1 mutations

  • Ex vivo analysis of primary cells from different tissues

  • Systemic physiological measurements (exercise capacity, stress resistance)

• Translational disease models:

  • Patient-derived cells carrying mitofilin/FCJ1 variants

  • Disease models with secondary mitochondrial dysfunction

  • Aging-related studies focusing on progressive mitochondrial changes

  • Infectious disease models examining host-pathogen interactions involving FCJ1

• Computational integration:

  • Molecular dynamics simulations connecting structure to membrane interactions

  • Mathematical modeling of how structural alterations propagate to functional outcomes

  • Network analysis linking FCJ1 to broader cellular pathways

  • Predictive models of physiological consequences based on structural perturbations

This integrative approach would help researchers understand how the molecular details of FCJ1 structure and interactions, particularly involving the critical C-terminal domain, translate into mitochondrial functional properties and ultimately affect cellular and organismal physiology in both normal and disease states . The connection between FCJ1's interaction with the TOB/SAM complex and its role in stabilizing crista junctions represents a particularly important example of how structural insights can inform understanding of mitochondrial functional architecture.

What are the most reliable commercial sources and quality control considerations for obtaining recombinant Ajellomyces capsulata FCJ1 protein?

When selecting commercial sources for recombinant Ajellomyces capsulata FCJ1 protein, researchers should consider reliability, quality control measures, and product specifications:

Recommended commercial sources:

Multiple suppliers offer recombinant FCJ1 proteins with varying specifications. Based on available information, reliable options include:

• CreativeBiomart - Offers recombinant full-length Ajellomyces capsulata FCJ1 protein with His-tag, expressed in E. coli, with >90% purity by SDS-PAGE .

• GeneBioSystems - Provides recombinant Ajellomyces capsulata FCJ1 with various tag options and specific amino acid sequence verification .

Critical quality control considerations:

• Protein integrity verification:

  • Confirmation of expected molecular weight (~75-80 kDa for full-length protein)

  • Verification of N-terminal and C-terminal sequence integrity

  • Mass spectrometry validation of complete amino acid sequence

  • Western blot with domain-specific antibodies

• Functional activity assessment:

  • Oligomerization capacity testing

  • Binding assays with known interaction partners

  • Conformational analysis by circular dichroism

  • Thermal stability measurements

• Storage and handling specifications:

  • Proper reconstitution in recommended buffers (typically Tris-based, pH 8.0)

  • Addition of stabilizers like glycerol (recommended 50% final concentration)

  • Aliquoting to avoid freeze-thaw cycles

  • Storage recommendations (-20°C/-80°C for long-term; 4°C for working stocks)

• Expression system considerations:

  • Bacterial expression may lack eukaryotic post-translational modifications

  • Tag position and type (His, GST, etc.) may affect certain interactions

  • Expression in fungal systems might provide more native-like protein

  • Endotoxin testing for proteins expressed in bacterial systems

When obtaining recombinant FCJ1, researchers should always verify the product specification details, including the exact amino acid range expressed (full-length mature protein typically spans residues 43-685 or 43-686), the position and type of affinity tags, and the buffer composition, as these factors can significantly influence experimental outcomes .

What experimental resources are essential for laboratories beginning research on FCJ1 and mitochondrial crista junction biology?

Laboratories initiating research programs focused on FCJ1 and crista junction biology should consider acquiring these essential experimental resources:

• Genetic tools and biological materials:

  • FCJ1 expression constructs with various tags and domain deletions

  • CRISPR/Cas9 constructs targeting FCJ1

  • FCJ1-deficient cell lines (if available) or knockout/knockdown systems

  • Antibodies recognizing different domains of FCJ1

  • Fluorescent protein fusions for live-cell imaging

• Imaging resources:

  • Access to transmission electron microscopy for ultrastructural analysis

  • Confocal or super-resolution microscopy for protein localization

  • Live-cell imaging systems with environmental control

  • Image analysis software for quantification of mitochondrial parameters

• Biochemical and molecular biology equipment:

  • Ultracentrifuges for membrane fractionation studies

  • Equipment for mitochondrial isolation and subfractionation

  • Protein interaction analysis tools (co-IP, pull-down assays)

  • Respirometry equipment to measure mitochondrial function

• Cell culture and model systems:

  • Fungal culture facilities if working with Ajellomyces/Histoplasma

  • Mammalian cell culture systems for comparative studies

  • Potential model organisms (yeast, flies, mice) with appropriate genetic tools

  • Primary cell isolation capabilities for tissue-specific studies

• Computational resources:

  • Software for structural analysis and modeling

  • Statistical packages for complex data analysis

  • Image processing tools for electron microscopy and fluorescence microscopy

  • Database access for literature and bioinformatic resources

• Specialized reagents and assays:

  • Membrane potential-sensitive dyes

  • Mitochondrial morphology markers

  • Tools for measuring reactive oxygen species

  • Assays for mitochondrial protein import and assembly