DCTN2 (1-406) Human

What is DCTN2 and what are its key cellular functions?

DCTN2, also known as dynamitin or p50, is a 50kDa subunit of dynactin, a macromolecular complex consisting of 10-11 subunits ranging in size from 22 to 150 kDa. DCTN2 is present in 4-5 copies per dynactin molecule and is comprised of 3 short alpha-helical coiled-coil domains . The protein plays crucial roles in multiple cellular functions, including:

• Anchoring microtubules to centrosomes

• Synapse formation during brain development

• ER-to-Golgi transport

• Centripetal movement of lysosomes and endosomes

• Spindle formation and chromosome movement

• Nuclear positioning

• Axonogenesis

Dynactin as a complex binds to both microtubules and cytoplasmic dynein, functioning as a crucial adaptor for dynein-mediated transport along microtubules .

What is the structural composition of DCTN2 (1-406) Human Recombinant Protein?

DCTN2 (1-406) Human Recombinant protein is produced in E. coli as a single, non-glycosylated polypeptide chain containing 429 amino acids (1-406 a.a. of native sequence) with a molecular mass of 47.2kDa . The recombinant protein is engineered with a 23 amino acid His-tag at the N-terminus to facilitate purification and detection . The complete amino acid sequence includes:

MGSSHHHHHH SSGLVPRGSH MGSMADPKYA DLPGIARNEP DVYETSDLPE DDQAEFDAFA QELEELTSTS VEHIIVNPNA AYDKFKDKRV GTKGLDFSDR IGKTKRTGYE SGEYEMLGEG LGVKETPQQK YQRLLHEVQE LTTEVEKIKT TVKESATEEK LTPVLLAKQL AALKQQLVAS HLEKLLGPDA AINLTDPDGA LAKRLLLQLE ATKNSKGGSG GKTTGTPPDS SLVTYELHSR PEQDKFSQAA KVAELEKRLT ELETAVRCDQ DAQNPLSAGL QGACLMETVE LLQAKVSALD LAVLDQVEAR LQSVLGKVNE IAKHKASVED ADTQSKVHQL YETIQRWSPI ASTLPELVQR LVTIKQLHEQ AMQFGQLLTH LDTTQQMIAN SLKDNTTLLT QVQTTMRENL ATVEGNFASI DERMKKLGK

How does DCTN2 contribute to the dynactin complex architecture?

The dynactin complex itself is a 1.4 MDa assembly consisting of multiple subunits that work together to regulate dynein activity. DCTN2's positioning within this complex enables it to participate in cargo recognition and binding specificity, contributing to the diverse cellular functions of the dynein-dynactin machinery .

What is the role of DCTN2 in viral infection pathways, particularly HIV-1?

DCTN2 plays a significant role in HIV-1 infection processes through its function in the dynein-dynactin complex. Research has demonstrated that depletion of dynactin components, including DCTN2, DCTN3, and ACTR1A, significantly decreased cell permissiveness to HIV-1 . The mechanistic basis involves:

• The dynein-dynactin-BICD2 complex is required for efficient HIV-1 infection and nuclear entry

• HIV-1 utilizes this complex for intracellular transport across the cytoplasm to reach the nucleus

• BICD2 functions as a capsid-specific adaptor that facilitates binding between the HIV-1 capsid and dynein

Validation experiments involving siRNA-mediated depletion of DCTN2 confirmed its importance in HIV-1 infection. Both pooled and individual siRNAs targeting DCTN2 showed similar inhibitory effects on HIV-1 infection, arguing against off-target effects . This suggests that DCTN2 represents a potential target for antiviral therapeutic strategies.

How can researchers effectively assess DCTN2 interactions with other components of the dynactin complex?

Researchers can employ several methodological approaches to study DCTN2 interactions with other dynactin components:

• Co-immunoprecipitation (Co-IP): Using antibodies against DCTN2 to pull down associated proteins, followed by immunoblotting for other dynactin components. This technique has been successfully used to demonstrate interactions between dynactin components and BICD2 adaptors .

• In vitro binding assays: Utilizing purified recombinant DCTN2 (1-406) Human protein to assess direct binding to other dynactin subunits under controlled conditions .

• Proximity ligation assays (PLA): Detecting protein-protein interactions in situ with high specificity and sensitivity.

• Cryo-electron microscopy: For structural analysis of DCTN2 within the dynactin complex, providing insights into interaction interfaces and conformational changes during complex assembly .

• Yeast two-hybrid screening: To identify novel interaction partners of DCTN2 within the complex.

When designing these experiments, researchers should consider including appropriate controls, such as mutant versions of DCTN2 with altered binding interfaces, to validate the specificity of observed interactions.

What experimental approaches are recommended for studying DCTN2's role in spindle formation and chromosome movement?

To investigate DCTN2's functions in spindle formation and chromosome movement, researchers can implement the following experimental approaches:

• Live-cell imaging with fluorescently tagged DCTN2: Enables real-time visualization of DCTN2 localization and dynamics during mitosis.

• siRNA-mediated knockdown or CRISPR-Cas9 knockout: Functional analysis through depletion of DCTN2 to assess effects on spindle formation and chromosome segregation. Previous studies have successfully used this approach to demonstrate dynein's role in HIV-1 infection .

• Dominant-negative mutant expression: Overexpression of dysfunctional DCTN2 variants can disrupt dynactin complex function, revealing DCTN2's specific contributions to mitotic processes.

• Immunofluorescence microscopy: To examine DCTN2 colocalization with spindle components during different mitotic phases.

• In vitro reconstitution assays: Using purified components including DCTN2 (1-406) Human recombinant protein to reconstitute aspects of spindle assembly and function .

A comprehensive experimental design should include appropriate controls and time-course analyses to capture dynamic processes throughout the cell cycle.

What are the optimal storage and handling conditions for DCTN2 (1-406) Human Recombinant Protein?

For optimal preservation of DCTN2 (1-406) Human Recombinant Protein activity and stability, researchers should follow these storage and handling guidelines:

• Long-term storage: Store at -20°C for extended periods. For very long-term storage, adding a carrier protein (0.1% HSA or BSA) is recommended to enhance stability .

• Short-term storage: After reconstitution, store at 4°C for usage within a few days .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces protein activity and should be minimized .

• Reconstitution conditions: The protein is typically supplied in a solution containing 20mM Tris-HCl (pH8.0), 20% glycerol, 0.15M NaCl, and 1mM DTT at a concentration of 0.5mg/ml .

• Working solution preparation: When preparing working dilutions, use buffers containing stabilizing agents such as BSA to prevent protein adsorption to tubes and loss of activity.

• Transport considerations: The protein is typically shipped with wet ice to maintain integrity during transport .

These conditions ensure maximum retention of structural integrity and functional activity of the recombinant protein for experimental applications.

What are the recommended protocols for validating DCTN2 (1-406) Human Recombinant Protein for functional studies?

Before using DCTN2 (1-406) Human in functional studies, researchers should validate both its purity and biological activity using the following protocols:

• Purity assessment:

  • SDS-PAGE analysis to confirm >90% purity as specified by manufacturers

  • Western blotting using anti-DCTN2 and anti-His-tag antibodies to verify protein identity

  • Mass spectrometry for precise molecular weight confirmation

• Functional validation:

  • Microtubule binding assays to confirm the protein's ability to interact with microtubules

  • Co-sedimentation assays with dynein components to verify interaction capability

  • ATPase activity assays if studying the impact on dynein motor function

• Structural integrity verification:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure

  • Size-exclusion chromatography to assess aggregation state and homogeneity

• Batch-to-batch consistency:

  • Comparison with previous lots using standardized functional assays

  • Documentation of lot-specific activity metrics for experimental normalization

These validation steps ensure that experimental outcomes reflect the true biological activity of DCTN2 rather than artifacts from protein degradation or inactivity.

How can researchers effectively use siRNA or CRISPR-Cas9 to study DCTN2 function in cellular models?

When using genetic perturbation approaches to study DCTN2 function, researchers should consider these methodological guidelines:

• siRNA-mediated knockdown:

  • Use pooled siRNAs targeting different regions of DCTN2 mRNA to enhance knockdown efficiency

  • Include multiple individual siRNAs separately to control for off-target effects

  • Validate knockdown efficiency using both RT-PCR for mRNA levels and immunoblot analysis for protein depletion

  • Consider timing of experiments, as knockdown effects may vary depending on protein half-life

• CRISPR-Cas9 knockout/knockdown:

  • Design multiple guide RNAs targeting early exons of DCTN2

  • Generate clonal cell lines and verify knockout by sequencing and immunoblotting

  • Consider creating conditional knockout systems (e.g., using Cre-loxP) due to potential lethality of complete DCTN2 knockout

  • For essential genes, consider CRISPR interference (CRISPRi) for tunable repression

• Phenotypic analysis:

  • Include comprehensive phenotypic assays covering multiple DCTN2 functions (e.g., microtubule organization, intracellular transport, cell division)

  • Perform rescue experiments with wild-type DCTN2 to confirm specificity of observed phenotypes

  • Consider time-course analyses to distinguish primary from secondary effects

• Controls:

  • Use non-targeting siRNAs or guide RNAs as negative controls

  • Include positive controls targeting genes with well-characterized phenotypes

  • For viral infection studies, include controls for cell viability to distinguish specific from non-specific effects

This approach has been successfully applied in HIV-1 studies, where validation experiments confirmed that observed effects were due to specific depletion of target proteins rather than off-target effects .

How can DCTN2 (1-406) Human be used in in vitro binding assays?

DCTN2 (1-406) Human recombinant protein can be effectively utilized in in vitro binding assays to study molecular interactions using the following approaches:

• Pull-down assays:

  • Leverage the N-terminal His-tag for immobilization on Ni-NTA resin

  • Incubate with potential binding partners from cell lysates or purified proteins

  • Analyze interactions by SDS-PAGE, western blotting, or mass spectrometry

  • Include appropriate controls (e.g., unrelated His-tagged proteins) to confirm specificity

• Surface Plasmon Resonance (SPR):

  • Immobilize DCTN2 (1-406) on sensor chips via His-tag

  • Measure real-time binding kinetics with potential interaction partners

  • Determine association and dissociation constants for quantitative analysis

• Microscale Thermophoresis (MST):

  • Label DCTN2 (1-406) with fluorescent dyes

  • Measure binding affinities in solution with minimal protein consumption

  • Suitable for studying interactions with large complexes like dynein

• Biomolecular complex reconstitution:

  • Combine DCTN2 (1-406) with other purified dynactin components

  • Study assembly dynamics and structural requirements

  • Assess impact of mutations or post-translational modifications on complex formation

These in vitro approaches allow researchers to dissect the molecular details of DCTN2 interactions with binding partners under controlled conditions, complementing cellular studies.

What are the implications of DCTN2 in neurodevelopmental and neurodegenerative diseases?

While the search results don't directly address DCTN2's role in neurodegenerative diseases, we can infer potential implications based on related information:

• Neurodevelopmental functions:

  • DCTN2 has a documented role in synapse formation during brain development

  • As part of the dynactin complex, it likely contributes to neuronal migration, axonal transport, and dendrite formation

• Connection to dyneinopathies:

  • Mutations in dynein heavy chain (DYNC1H1) cause various neurodegenerative diseases, including spinal muscular atrophy with lower extremity dominance (SMA-LED), congenital muscular dystrophy (CMD), and Charcot-Marie-Tooth disease (CMT)

  • As a key dynein regulator, DCTN2 dysfunction might contribute to similar pathologies

• Potential research directions:

  • Investigate DCTN2 expression patterns in neurodegenerative disease models

  • Examine genetic associations between DCTN2 variants and neurological disorders

  • Study the effects of DCTN2 dysfunction on axonal transport in neuronal cultures

  • Develop animal models with conditional DCTN2 knockout in neuronal populations

This represents an important area for future research, as components of the intracellular transport machinery are increasingly recognized as contributors to neurodegenerative processes.

What are the emerging research directions for DCTN2 (1-406) Human?

Several promising research directions for DCTN2 (1-406) Human are emerging:

• Therapeutic targeting in viral infections:

  • The established role of DCTN2 in HIV-1 infection suggests potential for developing antivirals targeting the dynein-dynactin-BICD2 interaction with viral capsids

  • Investigation of DCTN2's role in other viral infections could reveal common mechanisms

• Structural biology approaches:

  • High-resolution structural studies of DCTN2 within the dynactin complex could provide insights into function

  • Cryo-EM and X-ray crystallography with recombinant DCTN2 (1-406) Human could elucidate interaction interfaces

• Systems biology integration:

  • Network analysis of DCTN2 interactions within the cellular transport machinery

  • Computational modeling of dynein-dynactin complex formation and regulation

• Development of DCTN2-based research tools:

  • Engineered DCTN2 variants as molecular probes for intracellular transport

  • Biosensors based on DCTN2 to monitor dynactin complex assembly in live cells

• Cell-type specific functions:

  • Investigation of DCTN2's specialized roles in neurons, immune cells, and other cell types with unique transport requirements

  • Tissue-specific knockdown or knockout studies to identify context-dependent functions