Recombinant Danio rerio Dynamin-like 120 kDa protein, mitochondrial (opa1)

What is the function of Danio rerio OPA1 in mitochondrial dynamics?

Danio rerio OPA1, like its mammalian counterpart, functions primarily as a mitochondrial inner membrane remodeling protein with dual roles in maintaining mitochondrial morphology and energetics. It mediates inner membrane fusion following Mitofusin (Mfn)-mediated outer membrane fusion and maintains proper cristae structure essential for respiratory efficiency . Studies in zebrafish demonstrate that OPA1 is essential for proper mitochondrial metabolism during early embryonic development . The protein's function appears highly conserved among vertebrates, with zebrafish OPA1 knockdown disrupting mitochondrial morphology in a manner consistent with observations in mammalian systems .

How is OPA1 expressed during zebrafish development?

OPA1 expression in zebrafish shows dynamic regulation throughout embryonic development. Western blot analysis reveals that Danio rerio OPA1 exists as multiple isoforms, similar to other vertebrates . Specifically, detectable isoforms of approximately 100, 85, and 80 kDa, along with a doublet at approximately 78 kDa, show differential expression patterns across developmental stages . Interestingly, the larger isoforms gradually increase in abundance over time regardless of experimental manipulation, suggesting stage-specific regulations of different OPA1 variants during development . This temporal regulation pattern indicates the critical importance of specific OPA1 isoforms at different developmental stages.

What phenotypes result from OPA1 depletion in zebrafish embryos?

Morpholino-mediated knockdown of OPA1 in zebrafish embryos produces multiple developmental defects:

These phenotypes collectively demonstrate that OPA1 is essential for normal vertebrate development, particularly affecting high-energy-demanding tissues like the heart, nervous system, and developing visual structures .

How conserved is OPA1 between zebrafish and humans?

The OPA1 protein shows remarkably high conservation between zebrafish and humans, evident in both structure and function. This conservation is demonstrated by the successful detection of zebrafish OPA1 using a human OPA1 antibody in Western blot analysis, indicating substantial epitope preservation . Functionally, both human and zebrafish OPA1 are essential for proper mitochondrial fusion and cristae maintenance . Like human OPA1, zebrafish OPA1 exists as multiple isoforms resulting from alternative splicing and proteolytic processing . This high degree of conservation makes zebrafish an excellent model organism for studying fundamental aspects of OPA1 biology relevant to human health and disease.

What are the optimal methods for manipulating OPA1 expression in zebrafish?

For effective manipulation of OPA1 expression in zebrafish, several approaches have proven successful:

Translation-blocking morpholinos:

• Microinjection into 1-4 cell stage embryos delivers efficient knockdown

• Verification of knockdown effectiveness via Western blot analysis is essential

• A 5-bp mismatch control morpholino should be used to control for off-target effects

• Morpholino effectiveness typically diminishes by 96 hpf, as evidenced by partial recovery of protein levels

Temporal considerations:

• Analysis should be performed across multiple developmental timepoints (24, 48, 72, and 96 hpf)

• Yolk protein interferes with OPA1 protein migration in gels; removal of yolk cells prior to protein extraction improves results

• Different isoforms show varied susceptibility to knockdown, requiring comprehensive analysis of all bands

For advanced studies, CRISPR-Cas9 gene editing could provide more stable and specific genetic manipulation, though this approach was not specifically described in the search results.

How does OPA1 knockdown affect mitochondrial bioenergetics in zebrafish?

OPA1 depletion in zebrafish embryos causes significant bioenergetic alterations without compromising mitochondrial efficiency:

These findings differ notably from studies in OPA1 mutant fibroblasts, which typically show decreased RCR . This discrepancy likely reflects differences between isolated cell cultures and whole organisms with compensatory mechanisms . The increased RCR in OPA1-deficient zebrafish aligns with decreased basal respiration due to reduced ATP turnover/demand, suggesting a complex metabolic adaptation in the developing organism .

What structural features of OPA1 are critical for its membrane remodeling function?

Recent cryo-EM studies of human OPA1 have revealed critical structural elements that are likely conserved in zebrafish OPA1:

Key structural domains:

• Paddles Domain (PD): Adopts a unique hammer shape with dimensions of ~60Å × 40Å × 28Å, composed of four α-helices and three flexible loops

• Stalk Domain: Forms a tight helical bundle in assembled filaments

• Bundle Signalling Element (BSE): Connects the GTPase domain to the stalk

• GTPase Domain: Shows substantial conformational heterogeneity in assembled filaments

Critical interfaces:

• Interface 7: An intersubunit interface between membrane-interacting PDs, spanning a buried solvent-accessible surface area of approximately 300Ų

• Salt bridges between conserved Asp812 and Lys819 residues stabilize this interface

• K819E mutation completely abrogates membrane remodeling, resulting in severely fragmented mitochondria

Membrane interaction:

• The PD contains at least two membrane-interacting regions

• Assembled filaments can adopt different conformations with inner leaflet diameters ranging from 7 nm to 22 nm

• A "constricted" state with 7.6 nm inner lumen and an "expanded" state with 19.3 nm inner diameter have been observed

These structural insights provide a framework for designing targeted mutations in zebrafish OPA1 to investigate specific aspects of its function in membrane remodeling during development.

How do different isoforms of OPA1 contribute to mitochondrial function in zebrafish?

Zebrafish OPA1, like its mammalian counterpart, exists as multiple isoforms through alternative splicing and proteolytic processing . These include long membrane-anchored forms (L-OPA1) and short soluble forms (S-OPA1) . The differential expression and regulation of these isoforms during development suggest distinct functional roles:

Isoform characterization in zebrafish:

• Multiple isoforms observed at approximately 100, 85, 80 kDa, plus a 78 kDa doublet

• Larger isoforms increase in abundance over developmental time

• The 78 kDa doublet shows unique regulation patterns, increasing to four times the control level at 48 hpf following morpholino treatment

Functional implications:

• Based on mammalian studies, L-OPA1 likely anchors to the inner membrane while S-OPA1 exists as a soluble form

• The balance between these forms likely regulates mitochondrial fusion and cristae maintenance

• S-OPA1 has been shown to form helical assemblies that can remodel membranes with diameters ranging from 7 nm to 22 nm

While zebrafish-specific isoform functions were not directly characterized in the search results, the conservation between human and zebrafish OPA1 suggests similar functional diversification of isoforms. Research investigating the specific roles of each isoform in zebrafish development would be valuable for understanding the evolutionary conservation of OPA1 regulation mechanisms.

How does OPA1 interact with other mitochondrial dynamics proteins in zebrafish?

Although zebrafish-specific interactions were not detailed in the search results, the high conservation of mitochondrial dynamics machinery suggests interactions similar to those observed in mammalian systems:

Expected interaction partners:

• Mitofusins (Mfn1/2): OPA1 likely works in concert with these outer membrane fusion proteins, as inner membrane fusion mediated by OPA1 follows Mfn-mediated outer membrane fusion

• DLP1 (Drp1): This mitochondrial fission protein may function antagonistically to OPA1-mediated fusion

• Mff, MiD49, MiD51: These DLP1 receptors may indirectly affect OPA1 function by regulating fission events

• Cardiolipin (CL): This mitochondrial phospholipid likely activates OPA1 GTPase activity in zebrafish as it does in other species

• Phosphatidic acid (PA): May create a lipid environment favorable for fusion

Regulatory relationships:

Research specifically examining these interactions in zebrafish would provide valuable insights into the evolutionary conservation of mitochondrial dynamics regulatory networks.

What are the best approaches for measuring OPA1-related phenotypes in zebrafish?

Based on the search results, several methodological approaches have proven effective for characterizing OPA1-related phenotypes in zebrafish:

Morphological assessment:

• Standard morphological measurements (body length, eye size) at multiple timepoints (24, 48, 72, 96 hpf)

• Visual observation of circulation patterns and blood pooling

• Documentation of edema in pericardium, yolk, and hindbrain ventricle

Functional assessments:

• Heart rate quantification

• Behavioral analysis including startle response and locomotor activity measurements

• Yolk utilization as an indicator of metabolic function

Bioenergetic analysis:

• Oxygen consumption rate (OCR) measurements with inhibitor treatments to determine:

  • Basal respiration

  • ATP turnover

  • Proton leak

  • Maximal respiratory capacity

  • Respiratory control ratio

Molecular characterization:

• Western blot analysis of OPA1 isoforms (requires yolk removal)

• Potential mitochondrial morphology assessment (mentioned but not detailed in results)

These methods should be performed at multiple developmental timepoints (24, 48, 72, and 96 hpf) to capture the dynamic nature of OPA1's role during development.

How can zebrafish models inform human OPA1-related disease mechanisms?

Zebrafish OPA1 models offer several advantages for investigating human disease mechanisms:

Translational insights:

• High conservation of OPA1 structure and function between species enables direct comparisons

• The optical transparency of zebrafish embryos allows real-time visualization of developmental processes affected by OPA1 dysfunction

• Whole-organism effects reveal systemic consequences of OPA1 deficiency not observable in cell culture

Limitations and considerations:

• Differences in RCR responses between zebrafish and human cell models highlight species-specific compensatory mechanisms

• Temporary knockdown (morpholino) versus stable genetic mutations may yield different phenotypes

• Some human disease aspects (progressive optic atrophy) may develop over longer timeframes than standard zebrafish experimental periods

Potential applications:

• Testing candidate therapeutics for OPA1-related diseases in a whole-organism context

• Structure-function studies based on human disease mutations introduced into the zebrafish ortholog

• Investigation of tissue-specific vulnerabilities to OPA1 dysfunction

The zebrafish model's greatest strength lies in its ability to bridge cellular mechanistic studies with whole-organism physiology, providing unique insights into OPA1-related disease progression and potential interventions.

What are the most promising approaches for therapeutic targeting of OPA1 dysfunction?

Based on the understanding of OPA1 structure and function from the search results, several therapeutic strategies could be explored:

Structure-guided approaches:

• Small molecules targeting critical interfaces such as Interface 7 or the PD-membrane interactions might modulate OPA1 activity

• Peptide mimetics of key OPA1 domains could potentially stabilize OPA1 assemblies or prevent pathological associations

• Lipid-based therapies targeting cardiolipin or phosphatidic acid levels might enhance remaining OPA1 function

Gene therapy approaches:

• Supplementation with functional OPA1 in haploinsufficient conditions

• Correction of specific mutations using CRISPR-based strategies

• Modulation of OPA1 processing to alter L-OPA1/S-OPA1 ratios

Metabolic interventions:

• Compounds enhancing mitochondrial biogenesis to compensate for OPA1 dysfunction

• Targeting compensatory pathways identified in the zebrafish model with increased RCR despite OPA1 depletion

The zebrafish model provides an excellent platform for initial screening of such therapeutic approaches, offering whole-organism assessment of efficacy and toxicity.

What unexplored aspects of OPA1 biology warrant further investigation?

Several knowledge gaps regarding OPA1 biology emerge from the search results:

Isoform-specific functions:

• The functional significance of multiple zebrafish OPA1 isoforms and their developmental regulation

• Mechanisms controlling the varying susceptibility of different isoforms to knockdown

• The unexpected increase in the 78 kDa doublet at 48 hpf in morphants

Tissue-specific requirements:

• Why certain tissues (eyes, heart, brain) are particularly affected by OPA1 deficiency

• Cell-type specific OPA1 expression patterns during development

• Compensatory mechanisms operating in different tissues

Mechanistic questions:

Evolutionary aspects:

• Comparative analysis of OPA1 function across vertebrate models

• Conservation of regulatory mechanisms controlling OPA1 activity

• Species-specific adaptations in mitochondrial dynamics machinery

These unresolved questions represent promising avenues for future research using the zebrafish model in conjunction with structural and biochemical approaches.