Recombinant Xenopus laevis Torsin-4A-B (tor4a-b)

What is Recombinant Xenopus laevis Torsin-4A-B (tor4a-b) protein?

Recombinant Xenopus laevis Torsin-4A-B (tor4a-b) is a full-length (418 amino acids) protein expressed in E. coli with an N-terminal His-tag. The protein corresponds to the UniProt ID Q3KQ18 and is also known as Torsin family 4 member A-B. Its complete amino acid sequence is: MEESESQAPVPPHGISLVSSPVRAVIRIRRKIRTMKKSRLQVDLTGERSLDSAKASLRRQISMDRASLFKSSTYEKQQYFNFDTPTLEKLALTSQIRKRNRNKSRHVLYPGNVRKCLPVEQKSKAKRCLLLFVGIVCFQIFNAIENLDDNLQKYDLDGLEKTLQREVFGQTRAIEKLMDHLKDYLATHYHNKPLVLSFNGPSGVGKSHTGRLLAKHFRSVVDNDFVLQYYTNHNCPNESDVIQCQAEVSAMISQMISRAEIEEKIPVFLFDEVEAMPVALLDVLHSYFQLNQSNEYLNVVYILISNIGGHEITKFVLQNVSNDFFNLPQELHQIVLSSLRKHHSLWDVAEIVPFTLLEKRHILDCFLDELLREGFYPDHSNIESLAGQLRYYIKGNKEFSISGCKQVVAKVNLLQPYT .

How should Recombinant Xenopus laevis tor4a-b protein be reconstituted and stored for optimal stability?

For optimal stability and activity, reconstitute the lyophilized tor4a-b protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Add glycerol to a final concentration of 5-50% (with 50% being standard) to prevent freeze-thaw damage. Following reconstitution, store working aliquots at 4°C for up to one week, and store long-term aliquots at -20°C or preferably -80°C. Avoid repeated freeze-thaw cycles as these significantly degrade protein quality and function. Prior to opening, briefly centrifuge the vial to ensure all content is at the bottom .

What is the relationship between Torsin-4A-B and other Torsin family proteins?

Torsin-4A-B belongs to the Torsin family of AAA+ ATPases, which includes TorsinA, implicated in DYT1 dystonia. Unlike TorsinA, which is primarily involved in nuclear envelope-related functions, Torsin-4A-B's specific roles are less characterized. Torsin proteins generally function as molecular chaperones involved in protein quality control and cellular stress responses. Dysfunction of Torsin family proteins, particularly TorsinA, is associated with nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis, leading to the formation of aberrant FG-nucleoporin condensates . While they share structural similarities as AAA+ ATPases, Torsin family members may have distinct tissue expression patterns and cellular functions that warrant comparative studies.

How can Recombinant Xenopus laevis tor4a-b be used in developmental biology research?

In developmental biology research, Recombinant Xenopus laevis tor4a-b can be used to investigate protein function during embryonic development. Researchers can:

• Perform protein-protein interaction studies to identify binding partners during specific developmental stages

• Conduct overexpression experiments by microinjecting the recombinant protein into Xenopus embryos at the 1-2 cell stage

• Create loss-of-function models using morpholino technology targeting endogenous tor4a-b, followed by rescue experiments with the recombinant protein

• Combine with in situ hybridization techniques to correlate protein function with spatial expression patterns

Xenopus laevis is particularly advantageous for these studies due to its external development, transparent albino tadpole stage, and ease of access to the developing central nervous system for direct observation .

What imaging techniques are most effective for tracking tor4a-b function in Xenopus laevis?

For tracking tor4a-b function in Xenopus laevis, several advanced imaging techniques have proven effective:

• In vivo time-lapse imaging: Using fluorescently tagged tor4a-b or antibodies against tor4a-b combined with transparent albino tadpoles allows for direct observation of protein localization and dynamics in living embryos .

• Confocal microscopy of fixed specimens: After fixation in 4% paraformaldehyde, embryos can be processed for immunohistochemistry using antibodies against tor4a-b and related proteins.

• Transgenic reporter systems: Utilizing transgenic lines such as sox10-GFP (Xla.Tg(sox10:GFP)Jpsj) combined with tor4a-b manipulation can reveal effects on neural crest development and other processes .

• Clearing techniques: For deeper tissue imaging, hydrogen peroxide and formamide treatment followed by glycerol clearing enhances visualization of internal structures after tor4a-b manipulation .

These methods are particularly useful for investigating potential roles of tor4a-b in neural development, as the protein may share functional similarities with other Torsin family members involved in neurological processes.

How might tor4a-b function relate to nuclear envelope biology and disease models?

Based on the known functions of Torsin family proteins, tor4a-b may play significant roles in nuclear envelope biology that could be relevant to disease modeling. Research approaches should consider:

• Nuclear envelope integrity assessment: Investigate whether tor4a-b, like TorsinA, influences nuclear envelope structure and nuclear pore complex biogenesis. Xenopus embryos provide an excellent model for studying these processes due to rapid cell divisions and accessible visualization .

• Protein condensate formation: Examine if tor4a-b affects the formation or regulation of FG-nucleoporin condensates that can contribute to proteotoxic stress when dysregulated. This can be tested through co-immunoprecipitation studies with nuclear pore complex components and chaperone proteins like HSPA1A or DNAJB6 .

• Interaction with MLF2: Investigate potential interactions between tor4a-b and MLF2, which is known to modulate proteotoxic condensates in the context of Torsin dysfunction. Radioimmunoprecipitation assays could reveal whether tor4a-b participates in similar protein quality control networks .

• Ubiquitin-proteasome system: Assess tor4a-b's potential role in regulating protein degradation pathways, particularly for short-lived ubiquitinated proteins that can become stabilized in the context of nuclear envelope abnormalities .

What approaches can be used to study tor4a-b in the context of cell type-specific expression patterns?

To study tor4a-b in a cell type-specific manner, researchers can employ several sophisticated approaches:

• Transgenic driver systems: Utilize GAL4/UAS or CRE/loxP systems available in Xenopus to achieve cell type-specific manipulation of tor4a-b. For temporal control, heat shock promoters (hsp70) or tet-on systems with doxycycline treatment can be employed .

• CRISPR/Cas9 knockin strategies: Generate fluorescent reporter lines by knocking in fluorescent tags at the endogenous tor4a-b locus to observe native expression patterns without overexpression artifacts.

• Single-cell RNA sequencing: Apply scRNA-seq to identify cell populations with high tor4a-b expression and correlate with cell states or developmental trajectories.

• Conditional expression systems: Employ pSox2-bd::FP-like constructs that rely on endogenous transcription factors to drive expression in specific cell populations, such as neural progenitors .

These approaches allow for precise manipulation and observation of tor4a-b function in specific cell types and developmental contexts.

What controls should be included when studying tor4a-b function in Xenopus embryos?

When designing experiments to study tor4a-b function in Xenopus embryos, the following controls are essential:

Additionally, for antibody-based studies, researchers should validate antibody specificity by observing increased labeling in embryos injected with Xenopus tor4a-b mRNA in one cell at the 2-cell stage, comparing injected versus uninjected halves of the same embryo .

What are the optimal conditions for evaluating tor4a-b protein-protein interactions in Xenopus systems?

For robust evaluation of tor4a-b protein-protein interactions in Xenopus systems, consider the following methodological approach:

• Sample preparation: Harvest embryos at stage 26 (or appropriate developmental stage), wash in 50mM Tris buffer, and store at -80°C until processing. Prepare extracts under conditions that preserve native protein complexes .

• Co-immunoprecipitation: Use anti-His antibodies to pull down the His-tagged recombinant tor4a-b protein along with its binding partners. Alternatively, use antibodies against suspected interaction partners.

• Radioimmunoprecipitation: For detecting weaker or transient interactions, consider radioimmunoprecipitation approaches similar to those used for studying HSPA1A interactions. This approach successfully identified a 28 kDa band corresponding to MLF2 in TorsinA studies .

• Western blotting validation: Confirm interactions by western blotting, looking for enrichment of specific bands in experimental versus control conditions.

• Comparative analysis: Compare protein interaction profiles between normal conditions and developmental or stress contexts to identify condition-specific interactions.

The optimal buffer conditions would maintain native protein folding while allowing for specific binding detection, typically including:

• Physiological pH (7.4-7.6)

• 150mM NaCl

• Mild detergents (0.1% NP-40 or Triton X-100)

• Protease and phosphatase inhibitors

How can researchers distinguish between tor4a-b-specific effects and general AAA+ ATPase dysfunction?

Distinguishing tor4a-b-specific effects from general AAA+ ATPase dysfunction requires careful experimental design:

• Domain-specific mutations: Compare phenotypes resulting from mutations in tor4a-b-specific regions versus those in conserved AAA+ ATPase domains shared with other family members.

• Rescue experiments: Test whether other Torsin family members can rescue tor4a-b knockdown phenotypes. Partial rescue might indicate shared functions, while complete rescue would suggest redundancy.

• Substrate specificity assays: Identify substrates specifically recognized by tor4a-b but not other AAA+ ATPases through comparative binding assays and competition experiments.

• Structural analysis: Use the known amino acid sequence (MEESESQAPVPPHGISLVSSPVRAVIRIRRKIRTMKKSRLQVDLTGERSLDSAKASLRRQISMDRASLFKSSTYEKQQYFNFDTPTLEKLALTSQIRKRNRNKSRHVLYPGNVRKCLPVEQKSKAKRCLLLFVGIVCFQIFNAIENLDDNLQKYDLDGLEKTLQREVFGQTRAIEKLMDHLKDYLATHYHNKPLVLSFNGPSGVGKSHTGRLLAKHFRSVVDNDFVLQYYTNHNCPNESDVIQCQAEVSAMISQMISRAEIEEKIPVFLFDEVEAMPVALLDVLHSYFQLNQSNEYLNVVYILISNIGGHEITKFVLQNVSNDFFNLPQELHQIVLSSLRKHHSLWDVAEIVPFTLLEKRHILDCFLDELLREGFYPDHSNIESLAGQLRYYIKGNKEFSISGCKQVVAKVNLLQPYT) to identify unique structural features that might confer specific functions .

• Comparative expression analysis: Analyze tor4a-b expression patterns relative to other Torsin family members during development to identify unique spatiotemporal contexts for tor4a-b function.

What approaches can address contradictory results in tor4a-b functional studies?

When confronted with contradictory results in tor4a-b functional studies, researchers should systematically address potential sources of variation:

• Developmental timing precision: Ensure precise staging of embryos according to Nieuwkoop and Faber criteria, supplemented with hours post fertilization at standardized temperature (23°C) .

• Dosage effects: Test a range of morpholino or protein concentrations to identify potential threshold effects or biphasic responses.

• Genetic background effects: Compare results across different batches of Xenopus laevis embryos or consider using inbred lines to minimize genetic variability .

• Technical validation: Employ multiple independent techniques to assess the same endpoint:

  • Combine morpholino knockdown with CRISPR/Cas9 approaches

  • Validate protein loss through both western blotting and immunohistochemistry

  • Confirm phenotypes using both morphological and molecular readouts

• Context-dependent function: Consider whether contradictory results might reflect genuine biological context-dependency, such as tissue-specific or developmental stage-specific functions.

• Statistical rigor: Apply appropriate statistical analyses accounting for sample size, variability, and potential outliers. Consider power analyses to ensure adequate sample sizes for detecting biologically relevant effects.

How might single-cell approaches advance our understanding of tor4a-b function?

Single-cell approaches offer powerful new avenues to dissect tor4a-b function with unprecedented resolution:

• Single-cell RNA sequencing: Apply scRNA-seq to identify cell populations where tor4a-b is dynamically expressed during development, potentially revealing cell types most likely to be affected by tor4a-b manipulation.

• Single-cell proteomics: Emerging single-cell proteomics techniques could reveal cell-specific protein interaction networks involving tor4a-b.

• CRISPR screens at single-cell resolution: Develop cell type-specific CRISPR screens to identify genes that modify tor4a-b function in specific cellular contexts.

• Live imaging of protein dynamics: Combine the transparency of Xenopus embryos with fluorescently tagged tor4a-b to track protein dynamics at single-cell resolution during development .

• Spatial transcriptomics: Integrate spatial information with transcriptomic data to understand how tor4a-b function relates to tissue organization and cell-cell interactions.

These single-cell approaches could reveal heterogeneous responses to tor4a-b manipulation that might be masked in bulk analyses and provide insights into cell type-specific functions that could be relevant to understanding related human disorders.

What potential roles might tor4a-b play in protein quality control and neurological development?

Based on our understanding of Torsin family proteins and protein quality control mechanisms, tor4a-b may have several unexplored roles:

• Proteostasis regulation: Like other Torsins, tor4a-b might participate in protein quality control networks, potentially interacting with chaperones such as HSPA1A and DNAJB6 in a manner similar to what has been observed with TorsinA .

• Neural development: Given the involvement of Torsin family members in neurological disorders, tor4a-b may participate in processes critical for neural development. The Xenopus system offers excellent opportunities to investigate this through:

  • Visualization of neural progenitor proliferation and differentiation using transgenic lines

  • Assessment of effects on cell migration and morphogenesis during neural development

  • Evaluation of impacts on neuronal connectivity and circuit formation

• Nuclear envelope dynamics: Tor4a-b might regulate nuclear envelope organization and dynamics during rapid cell divisions that characterize embryonic development, potentially affecting nuclear pore complex biogenesis and nucleocytoplasmic transport .

• Stress response pathways: Investigation of tor4a-b regulation during cellular stress responses could reveal roles in proteotoxic stress management, particularly in the context of ubiquitin-proteasome system function .