Recombinant Danio rerio Dynactin subunit 2 (dctn2)

What is Dynactin subunit 2 (dctn2) and what is its primary function in zebrafish?

Dynactin subunit 2 (dctn2), also known as dynamitin or p50, is a 50 kDa intracellular phosphoprotein that plays a crucial role in stabilizing the dynactin complex. In zebrafish (Danio rerio), as in other vertebrates, four dctn2 molecules help form and stabilize the dynactin complex, which consists of 11 different proteins in total. This complex functions as an obligate cofactor for both dynein and kinesin motor proteins, which are essential for positioning the mitotic spindle during cell division and facilitating vesicular transport throughout the cell . The gene is officially designated as dynactin 2 (p50) in the zebrafish genome, with synonyms including dynamitin and zgc:63867, and has been assigned the Entrez Gene ID 394141 .

How is the dynactin complex structurally organized, and what is the specific role of dctn2 within this complex?

The dynactin complex is a multi-subunit structure composed of 11 different proteins that work together to regulate dynein-mediated transport. Within this complex, Dynactin subunit 2 (dctn2) serves as a critical stabilizing component. Specifically, four dctn2 molecules form a tetrameric structure that provides structural integrity to the dynactin complex . This tetrameric arrangement is essential for the proper assembly and function of the entire complex. The dctn2 subunit acts as a bridge between different components of the dynactin complex, allowing it to effectively interact with motor proteins. When the normal structure or expression of dctn2 is disrupted, the entire dynactin complex can become destabilized, potentially leading to defects in intracellular transport and other cellular processes dependent on motor protein function.

What are the most effective methods for expressing and purifying recombinant Danio rerio dctn2 protein?

For effective expression and purification of recombinant Danio rerio dctn2 protein, researchers typically employ bacterial expression systems, particularly E. coli, as demonstrated in comparable human DCTN2 expression protocols . The process begins with cloning the full-length dctn2 cDNA into an appropriate expression vector. The cDNA can be amplified from zebrafish total cDNA using specific primers targeting the dctn2 gene sequence .

A typical expression protocol involves:

• Transforming the expression construct into a suitable E. coli strain (BL21 or derivatives)

• Inducing protein expression with IPTG at optimal temperature (typically 18-25°C for complex proteins)

• Harvesting cells and lysing in a buffer containing appropriate detergents and protease inhibitors

• Purifying the protein using affinity chromatography (His-tag or GST-tag systems)

• Further purification through size exclusion or ion exchange chromatography if needed

• Formulating the purified protein in a stabilizing buffer similar to the formulation used for human DCTN2: 20mM Tris-HCl (pH8.0), 20% glycerol, 0.15M NaCl, and 1mM DTT

The final purified product should achieve greater than 90% purity as determined by SDS-PAGE analysis. For optimal stability, the protein should be stored at -20°C in the presence of glycerol .

How can researchers verify the functional activity of recombinant dctn2 in experimental systems?

Verifying the functional activity of recombinant dctn2 requires multiple approaches to confirm both its structural integrity and biological function. The following methodologies are recommended:

• Structural verification:

  • SDS-PAGE and Western blotting using specific antibodies against dctn2

  • Circular dichroism spectroscopy to confirm proper protein folding

  • Size exclusion chromatography to ensure proper oligomerization (tetramer formation)

• Functional assays:

  • In vitro binding assays with dynein and dynactin components to confirm complex formation

  • Microtubule co-sedimentation assays to verify interaction with microtubules

  • Cellular reconstitution experiments by introducing the recombinant protein into dctn2-depleted cells and assessing rescue of dynactin function

• Biological activity assessment:

  • Microinjection of recombinant dctn2 into dctn2-mutant zebrafish embryos to test for phenotypic rescue

  • Live cell imaging of fluorescently-tagged recombinant dctn2 to track incorporation into functional dynactin complexes

  • Assessment of axonal transport in motor neurons using fluorescent cargo tracking

Each of these methods provides complementary information about different aspects of dctn2 functionality, and combining multiple approaches yields the most comprehensive validation of the recombinant protein's activity.

What are the recommended protocols for studying dctn2 interactions with motor proteins in zebrafish models?

To study dctn2 interactions with motor proteins in zebrafish models, researchers can employ several complementary approaches:

• Genetic manipulation techniques:

  • Generate dctn2 mutant lines using CRISPR/Cas9 technology targeting the dctn2 gene

  • Create transgenic zebrafish expressing fluorescently tagged dctn2 and motor proteins for in vivo visualization

  • Develop conditional knockdown models using morpholinos or dominant-negative constructs

• Live imaging methods:

  • Perform in vivo axonal transport assays in single cells by microinjecting fluorescently labeled cargoes and tracking their movement

  • Use GCaMP calcium imaging in live, intact larvae to assess neuromuscular junction function in dctn2-modified models

  • Apply high-speed confocal microscopy to visualize motor protein-cargo complex movement in real-time

• Biochemical interaction studies:

  • Conduct co-immunoprecipitation assays using antibodies against dctn2 and motor proteins

  • Perform proximity ligation assays to detect protein-protein interactions in situ

  • Use yeast two-hybrid or split-GFP complementation assays to map interaction domains

• Structural analysis:

  • Apply cryo-electron tomography (cryo-ET) to visualize dynactin-motor protein complexes, similar to the techniques used for axonemal dynein studies in zebrafish

  • Combine with subtomographic averaging to achieve higher resolution of complex structures

These methods allow for comprehensive analysis of dctn2-motor protein interactions from molecular to organismal levels in the zebrafish model system.

How does dctn2 function in zebrafish neuronal development and what are the implications for studying neurodegenerative diseases?

Dynactin subunit 2 (dctn2) plays critical roles in zebrafish neuronal development through its function in the dynactin complex, which regulates retrograde axonal transport and other dynein-mediated processes. Research indicates that proper dctn2 function is essential for motor neuron development, axonal transport, and neuromuscular junction stability .

The study of dynactin components in zebrafish has significant implications for understanding neurodegenerative diseases, particularly those involving axonal transport defects. While much research has focused on Dynactin1 (DCTN1), whose reduced levels have been observed in sporadic amyotrophic lateral sclerosis (ALS) patients , the role of dctn2 in neurodegeneration is also relevant but less extensively characterized.

Research approaches to study dctn2 in neurodegeneration include:

• Creation of dctn2 depletion models in zebrafish to examine effects on:

  • Motor neuron axon outgrowth and pathfinding

  • Synapse formation and stability at neuromuscular junctions

  • Axonal transport dynamics of mitochondria, vesicles, and other cargoes

• Electrophysiological analysis of neuromuscular function in dctn2-deficient models, using techniques such as:

  • Paired motor neuron-muscle recordings to assess synapse functionality

  • GCaMP calcium imaging to visualize synaptic activity patterns

• Ultrastructural analysis of motor neurons and their synapses using:

  • Electron microscopy to examine synapse morphology and integrity

  • Cryo-electron tomography to visualize molecular transport complexes

These approaches can provide insights into how dctn2 dysfunction might contribute to neurodegenerative processes and potentially identify novel therapeutic targets for conditions like ALS, where dynactin complex impairments have been implicated in disease pathogenesis.

What is the relationship between dctn2 and axonemal dyneins in zebrafish cilia and flagella motility?

The relationship between dctn2 and axonemal dyneins in zebrafish cilia and flagella motility represents a complex interplay between different cellular transport systems. While dctn2 is primarily known for its role in the cytoplasmic dynactin complex that regulates cytoplasmic dynein, research suggests potential connections to the assembly and function of axonemal dyneins that power cilia and flagella motility.

In zebrafish, axonemal dyneins are organized in specific patterns within the axoneme, including outer arm dyneins (OADs) and several types of inner arm dyneins (IADs) . The proper assembly of these dyneins depends on dynein axonemal assembly factors (DNAAFs), including the PIH protein family. Studies have shown that mutations in PIH genes lead to defects in specific subtypes of axonemal dyneins, correlating with abnormal sperm motility and ciliary dysfunction .

The potential connection between dctn2 and axonemal dyneins may involve:

• Preassembly pathways: The dynactin complex (including dctn2) may participate in the cytoplasmic preassembly of axonemal dynein components before their transport to cilia/flagella.

• Differential expression patterns: Like the organ-specific expression patterns observed for dynein heavy chain (DNAH) genes in zebrafish , dctn2 may show tissue-specific expression that correlates with particular ciliary/flagellar functions.

• Transport functions: The dynactin complex could be involved in the intraflagellar transport of axonemal components, including dyneins, to the assembly sites within cilia and flagella.

Comparative analysis of dynein composition in different motile structures reveals organ-specific patterns, as shown in the following table based on DNAH gene expression:

Understanding these complex relationships provides insights into the fundamental mechanisms of cellular motility and transport systems in vertebrates.

How do mutations in dctn2 affect intracellular transport in different zebrafish cell types, and what methods best characterize these defects?

Mutations in dctn2 can have profound and cell type-specific effects on intracellular transport in zebrafish, reflecting the diverse roles of the dynactin complex in different cellular contexts. The impact of dctn2 mutations varies based on the specific transport requirements of each cell type, with neurons, muscle cells, and cells with motile cilia being particularly vulnerable.

Cell Type-Specific Effects:

• Neurons: Disruption of dctn2 function likely impairs retrograde axonal transport, potentially leading to:

  • Accumulation of cargoes at axon terminals

  • Reduced transport of signaling endosomes

  • Defective synaptic maintenance and neuromuscular junction stability

• Ciliated Epithelial Cells: In cells with motile cilia, such as those in Kupffer's vesicle, dctn2 mutations might affect:

  • Transport of components needed for cilia assembly and maintenance

  • Proper positioning of basal bodies

  • Ciliary beating patterns and fluid flow generation

• Muscle Cells: Effects may include:

  • Altered positioning of nuclei within the syncytial structure

  • Disrupted organization of the contractile apparatus

  • Compromised neuromuscular junction structure on the postsynaptic side

Methodologies for Characterizing Transport Defects:

For comprehensive characterization of transport defects resulting from dctn2 mutations, researchers should employ multiple complementary approaches:

• Live Imaging Techniques:

  • High-resolution confocal microscopy with fluorescently tagged cargo proteins

  • Spinning disk confocal microscopy for rapid acquisition of transport dynamics

  • Single-particle tracking of labeled vesicles or organelles

  • Photoactivation or photoconversion strategies to track specific subpopulations of proteins

• Functional Assays:

  • Electrophysiological recordings to assess synaptic transmission

  • Calcium imaging using GCaMP reporters to visualize activity patterns

  • Behavioral assays to correlate cellular defects with organismal phenotypes

• Structural Analysis:

  • Electron microscopy to examine ultrastructural changes

  • Cryo-electron tomography to visualize molecular complexes at high resolution

  • Immunohistochemistry to track accumulation of specific cargoes

• Biochemical Approaches:

  • Subcellular fractionation to assess protein localization

  • Western blotting to quantify changes in protein levels and modifications

  • Mass spectrometry to identify altered protein interactions

By combining these approaches, researchers can develop a comprehensive understanding of how dctn2 mutations affect transport processes in different cellular contexts and potentially identify therapeutic interventions for related human disorders.

How do the functions of dctn2 in zebrafish compare with those in other model organisms, and what are the implications for translational research?

The functions of dctn2 demonstrate both conservation and divergence across vertebrate and invertebrate model organisms, providing valuable comparative insights for translational research. Understanding these similarities and differences is crucial for effectively utilizing zebrafish as a model for human diseases involving dynactin dysfunction.

Comparative Function Across Model Organisms:

Implications for Translational Research:

• Disease Modeling: Zebrafish dctn2 models can provide insights into human conditions involving dynactin dysfunction, particularly neurodegenerative disorders like ALS where dynactin components have been implicated .

• Therapeutic Development: The conservation of core mechanisms makes zebrafish suitable for screening compounds that modulate dynactin function, potentially identifying therapeutics for human diseases.

• Developmental Disorders: The role of dctn2 in zebrafish development illuminates potential mechanisms of developmental abnormalities in humans with dynactin mutations.

• Organ-Specific Functions: The tissue-specific expression patterns of dynein genes in zebrafish suggest that dynactin may have organ-specific functions relevant to understanding tissue-specific manifestations of human diseases.

• Evolutionary Insights: Comparative analysis across species reveals which aspects of dynactin function are fundamental versus those that represent species-specific adaptations.

By integrating findings from multiple model systems, researchers can develop a more comprehensive understanding of dctn2 function applicable to human health and disease.

What are the latest techniques for studying dctn2 interactions with the cytoskeleton, and how might these advance our understanding of cellular dynamics?

Recent technological advances have significantly enhanced our ability to study dctn2 interactions with the cytoskeleton, providing unprecedented insights into cellular dynamics. These cutting-edge approaches span from single-molecule techniques to whole-organism imaging strategies.

Advanced Imaging Techniques:

• Super-Resolution Microscopy:

  • Structured illumination microscopy (SIM) to visualize dctn2-cytoskeleton interactions beyond the diffraction limit

  • Stochastic optical reconstruction microscopy (STORM) for nanometer-scale localization of individual dctn2 molecules

  • Stimulated emission depletion (STED) microscopy to observe dynamic interactions in live cells

• Single-Molecule Imaging:

  • Single-particle tracking to follow individual dctn2-containing complexes along cytoskeletal tracks

  • High-speed atomic force microscopy (HS-AFM) to visualize structural changes during interactions

  • Fluorescence resonance energy transfer (FRET) to detect direct molecular interactions in real-time

• Cryo-Electron Microscopy:

  • Cryo-electron tomography (cryo-ET) to visualize dctn2-containing complexes in their native cellular environment

  • Subtomographic averaging to achieve higher resolution of molecular complexes

  • Correlative light and electron microscopy (CLEM) to combine functional and structural information

Molecular Manipulation Approaches:

• Optogenetic Tools:

  • Light-inducible dimerization systems to control dctn2 recruitment to specific cellular locations

  • Optogenetic control of motor protein activity to study downstream effects on dctn2 function

• Genome Engineering:

  • CRISPR/Cas9-mediated tagging of endogenous dctn2 with fluorescent proteins

  • Creation of conditional knockout systems to study tissue-specific functions

  • Base editing to introduce specific point mutations corresponding to human disease variants

• Biochemical Reconstitution:

  • In vitro reconstitution of dctn2-containing complexes on artificial cytoskeletal structures

  • Microfluidic systems to study force generation and cargo transport

  • DNA origami scaffolds to precisely position dctn2 relative to cytoskeletal elements

Potential Advances in Understanding Cellular Dynamics:

These techniques are poised to advance our understanding of cellular dynamics in several key areas:

• Mechanistic Insights: Revealing how dctn2 contributes to the coordination between different motor proteins (dyneins and kinesins) during bidirectional transport.

• Regulatory Networks: Identifying the signaling pathways that modulate dctn2-cytoskeleton interactions in response to cellular needs.

• Structural Dynamics: Understanding conformational changes in the dynactin complex during its interaction with the cytoskeleton and how these relate to function.

• Developmental Processes: Elucidating how dctn2-dependent transport contributes to tissue patterning and morphogenesis in developing zebrafish.

• Disease Mechanisms: Clarifying how mutations or dysregulation of dctn2 lead to cellular pathology in neurodegenerative and developmental disorders.

By integrating these advanced techniques, researchers can build a comprehensive model of how dctn2 functions within the complex landscape of cytoskeletal dynamics and intracellular transport.

What are the potential applications of recombinant dctn2 in developing targeted therapies for cytoskeletal and motor protein-related disorders?

Recombinant dctn2 holds significant promise for developing targeted therapies for cytoskeletal and motor protein-related disorders, particularly neurodegenerative conditions where dynactin dysfunction has been implicated. The therapeutic potential spans from fundamental research tools to direct therapeutic interventions.

Therapeutic Potential of Recombinant dctn2:

• Replacement Therapy Approaches:

  • Direct protein replacement using modified recombinant dctn2 with enhanced cellular penetration

  • Gene therapy vectors expressing functional dctn2 for targeted delivery to affected tissues

  • mRNA-based therapies to transiently boost dctn2 expression in deficient cells

• Structural Correction Strategies:

  • Engineered dctn2 variants designed to stabilize mutant dynactin complexes

  • Peptide mimetics that recreate essential interaction domains of dctn2

  • Small molecules identified through structure-based drug design to stabilize dctn2-dependent complexes

• Diagnostic and Research Applications:

  • Biomarkers for dynactin-related disorders using recombinant dctn2 in binding assays

  • High-throughput screening platforms with recombinant dctn2 to identify therapeutic compounds

  • In vitro transport assays using recombinant components to evaluate potential therapeutic interventions

Specific Disease Applications:

• Neurodegenerative Disorders:

  • Amyotrophic Lateral Sclerosis (ALS): Where dynactin components have been directly implicated

  • Hereditary Spastic Paraplegia: Involving defects in axonal transport

  • Huntington's Disease: Where vesicular trafficking defects contribute to pathology

• Ciliopathies:

  • Primary Ciliary Dyskinesia: Potentially targeting the relationship between cytoplasmic transport and axonemal dynein assembly

  • Polycystic Kidney Disease: Addressing ciliary formation and maintenance defects

• Cancer Therapeutics:

  • Targeting mitotic spindle defects in cancer cells

  • Modulating cell migration through dynactin-dependent processes

Challenges and Future Directions:

Several challenges must be addressed to realize the therapeutic potential of recombinant dctn2:

• Delivery and Stability:

  • Development of effective blood-brain barrier penetration strategies for neurological applications

  • Engineering enhanced stability and half-life of recombinant dctn2 in physiological conditions

  • Targeted delivery systems to reach specific affected cell populations

• Functional Specificity:

  • Designing variants that correct specific pathogenic mechanisms without disrupting normal function

  • Understanding tissue-specific requirements for dctn2 to avoid off-target effects

  • Temporal control of therapeutic intervention to match developmental or disease stage

• Combination Approaches:

  • Integration with other therapies targeting complementary aspects of cytoskeletal dysfunction

  • Personalized medicine approaches based on specific mutations or disease mechanisms

  • Preventive strategies for individuals with genetic predispositions to dynactin-related disorders

The development of recombinant dctn2-based therapies represents a promising frontier in addressing currently incurable conditions involving cytoskeletal and motor protein dysfunction, with zebrafish models providing an excellent system for preclinical evaluation of these approaches.

What are the most promising future research directions for Danio rerio dctn2 studies?

The study of Danio rerio dctn2 stands at an exciting intersection of cellular biology, developmental science, and disease modeling. Several promising research directions emerge from current knowledge and technological capabilities:

• Integrative Multi-omics Approaches:

  • Combining proteomics, transcriptomics, and metabolomics to understand the broader cellular impact of dctn2 dysfunction

  • Mapping the complete interactome of dctn2 in different cellular contexts and developmental stages

  • Employing spatial transcriptomics to correlate dctn2 function with tissue-specific gene expression patterns

• Advanced In Vivo Imaging:

  • Developing zebrafish lines with endogenously tagged dctn2 using CRISPR knock-in strategies

  • Applying light-sheet microscopy for whole-organism imaging of dctn2 dynamics during development

  • Implementing intravital microscopy to observe dctn2 function in specific tissues within live animals

• Functional Diversification Analysis:

  • Investigating tissue-specific roles of dctn2 through conditional knockout approaches

  • Exploring potential differential functions of dctn2 splice variants

  • Examining evolutionary adaptations of dctn2 function across vertebrate species

• Therapeutic Development Platforms:

  • High-throughput screening for compounds that modulate dctn2 function or stability

  • Gene therapy approaches targeting dctn2 expression in disease models

  • Development of synthetic biology tools to engineer modified dynactin complexes with enhanced properties

• Systems Biology Integration:

  • Computational modeling of dynactin-dependent transport processes

  • Network analysis connecting dctn2 function to broader cellular pathways

  • Predictive modeling of how dctn2 mutations affect cellular function across different tissues

These research directions hold tremendous potential for advancing our understanding of fundamental cellular processes and developing novel therapeutic approaches for human diseases involving dynactin dysfunction.

How can researchers effectively collaborate and share resources in the field of dynactin research using zebrafish models?

Effective collaboration and resource sharing are essential for advancing dynactin research using zebrafish models. The following strategies can facilitate productive scientific exchange and accelerate progress in this field:

• Standardized Protocols and Reagents:

  • Establish community-wide standards for recombinant protein production and characterization

  • Develop validated protocols for assessing dynactin function in different zebrafish tissues

  • Create repositories for sharing validated antibodies, constructs, and zebrafish lines

• Collaborative Research Networks:

  • Form interdisciplinary consortia bringing together experts in protein biochemistry, developmental biology, and disease modeling

  • Establish regular workshops or conferences focused on dynactin research in zebrafish

  • Develop multi-institutional collaborations to leverage complementary expertise and resources

• Data Sharing Platforms:

  • Contribute to zebrafish-specific databases like ZFIN with standardized annotations for dynactin-related phenotypes

  • Establish open repositories for imaging data, particularly for resource-intensive techniques like cryo-ET

  • Develop shared bioinformatic pipelines for analyzing dynactin-related datasets

• Technology Transfer:

  • Organize practical workshops to disseminate specialized techniques like paired electrophysiological recordings or cryo-ET

  • Create video protocols and detailed methodological publications for complex procedures

  • Establish visiting scientist programs to facilitate direct transfer of technical expertise

• Coordinated Resource Development:

  • Collaborate on the generation of comprehensive toolkits (antibodies, transgenic lines, etc.)

  • Establish centralized facilities for specialized techniques like cryo-ET that require significant infrastructure

  • Coordinate the creation of mutant collections covering all components of the dynactin complex

By implementing these collaborative approaches, researchers can more effectively leverage the strengths of the zebrafish model system and accelerate progress in understanding dynactin biology and its implications for human health and disease.

What are the essential resources, databases, and tools for researchers studying Danio rerio dctn2?

Researchers studying Danio rerio dctn2 should be familiar with an array of specialized resources, databases, and tools that facilitate experimental design, data analysis, and integration with existing knowledge. The following comprehensive list provides essential resources for investigating dynactin biology in zebrafish:

Genomic and Sequence Resources:

• ZFIN (Zebrafish Information Network): The central repository for zebrafish genetic, genomic, and developmental data, containing detailed information on dctn2 expression patterns and mutant phenotypes.

• Ensembl Genome Browser: Provides genomic sequence, gene models, and comparative genomics for dctn2 across vertebrate species.

• NCBI Gene: Offers comprehensive gene information, including the dctn2 entry (Gene ID: 394141) with links to related sequences and literature .

• UniProt: Contains curated protein information for zebrafish dctn2, including functional annotations and domain structures.

Experimental Resources:

• ZIRC (Zebrafish International Resource Center): Supplies zebrafish lines, including potential dctn2 mutants and related strains.

• Addgene: Repository for plasmids and vectors useful for dctn2 studies, including expression constructs and CRISPR targeting vectors.

• The Zebrafish Book: Comprehensive guide to zebrafish husbandry and experimental techniques relevant to dctn2 research.

• CRISPRscan: Tool for designing effective guide RNAs for CRISPR/Cas9 editing of the dctn2 gene.

Structural and Interaction Data:

• Protein Data Bank (PDB): Contains structural information on dynactin components that can be used for modeling zebrafish dctn2.

• BioGRID: Database of protein-protein interactions that includes dynactin components.

• STRING: Provides predicted and known protein interaction networks for dctn2 and associated proteins.

• EMDB (Electron Microscopy Data Bank): Repository for EM data, including cryo-ET structures of dynactin-related complexes .

Expression and Phenotype Data:

• Expression Atlas: Contains RNA-seq and microarray data showing dctn2 expression across different tissues and developmental stages.

• ZFIN Expression Database: Specific resource for zebrafish gene expression patterns, including detailed information on dctn2.

• Phenotype Browser in ZFIN: Catalogues phenotypes associated with dctn2 mutations or knockdowns.

• Virtual Zebrafish Embryo: Interactive 3D atlas of zebrafish development useful for correlating dctn2 expression with anatomical structures.

Bioinformatics Tools:

• BLAST and HMMER: For sequence similarity searches to identify dctn2 homologs and domains.

• Clustal Omega: Multiple sequence alignment tool for comparing dctn2 across species.

• SMART and Pfam: Domain prediction tools for analyzing functional regions in dctn2.

• ZebrafishMine: Data mining tool integrating multiple data types for zebrafish genes, including dctn2.

Commercial Resources:

• GenScript: Provides cDNA ORF clones derived from zebrafish dctn2 .

• RayBiotech: Offers recombinant protein products similar to those needed for dctn2 research .

• R&D Systems: Supplies antibodies and related products for dynactin research .