Recombinant Xenopus laevis Rhophilin-2-A (rhpn2-a), partial

What is Rhophilin-2-A and what are its known functions in Xenopus laevis?

Rhophilin-2-A (rhpn2-a) is a GTP-Rho-binding protein in Xenopus laevis, identified with Uniprot number Q6DJJ6 . As a member of the rhophilin family, it functions as an effector protein for Rho GTPases, which are critical regulators of cytoskeletal dynamics, cell migration, and morphogenesis during embryonic development.

While specific functions in Xenopus have not been extensively characterized in the provided literature, researchers can investigate rhpn2-a function through:

• Gene expression analysis during developmental stages

• Protein localization studies using fluorescent tags

• Loss-of-function experiments using morpholinos or CRISPR-Cas9

• Binding assays with Rho family GTPases

These approaches allow for characterizing spatiotemporal expression patterns and potential developmental roles in amphibian models.

How should Recombinant Xenopus laevis Rhophilin-2-A be stored and handled for optimal stability?

Proper storage and handling of recombinant rhpn2-a are essential for maintaining protein integrity and activity. The recommended protocols include:

Storage conditions:

• Store at -20°C/-80°C for optimal stability

• Liquid form has approximately 6 months shelf life

• Lyophilized form maintains stability for approximately 12 months at -20°C/-80°C

Handling protocols:

• Briefly centrifuge vials 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 5-50% (preferably 50%) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Working aliquots may be stored at 4°C for up to one week

Repeated freezing and thawing should be avoided as it significantly reduces protein activity through denaturation and aggregation.

What expression systems are used for producing Recombinant Xenopus laevis Rhophilin-2-A?

The commercially available Recombinant Xenopus laevis Rhophilin-2-A is produced in mammalian cell expression systems . This approach offers several advantages for producing functionally active Xenopus proteins:

• Post-translational modifications similar to those in vertebrate systems

• Proper protein folding facilitated by mammalian chaperones

• Reduced endotoxin contamination compared to bacterial systems

• Higher probability of soluble protein expression

Alternative expression systems for Xenopus proteins include:

For studies requiring particularly high functional activity, expression in Xenopus embryos themselves can be considered, as demonstrated with other recombinant proteins in Xenopus models .

How can genetic code expansion techniques be applied to study Rhophilin-2-A function?

Genetic code expansion (GCE) provides powerful tools for studying protein function through site-specific incorporation of unnatural amino acids (UAAs) with novel chemical functionalities. This approach could be valuable for investigating rhpn2-a interactions and regulation.

Based on established protocols for Xenopus embryos, researchers can apply GCE to rhpn2-a studies through:

• Site-directed mutagenesis to introduce an amber stop codon (UAG) at positions of interest in the rhpn2-a gene

• Co-injection of pyrrolysyl-tRNA synthetase (PylRS) mRNA (250 pg) and pyrrolysyl tRNA (PylT, 7.5 ng) into one-cell stage Xenopus embryos

• Addition of UAAs through injection or media supplementation (optimal concentrations vary by UAA)

For rhpn2-a studies, particularly useful UAAs include:

• Photocaged lysines like 2 for temporal control of protein activity

• Azide-containing lysines like 5 for bioorthogonal chemistry applications

• Tetrazine-reactive lysines like 4 for protein-protein interaction studies

The efficiency of incorporation depends on several factors, including the UAA structure and the specific PylRS variant used. For phenylalanine-backbone UAAs, addition to embryo water (1 mM) has shown successful incorporation, while lysine-based UAAs typically require direct injection .

What are the recommended protocols for immunoprecipitation studies using Recombinant Xenopus laevis Rhophilin-2-A?

Immunoprecipitation (IP) studies are essential for investigating protein-protein interactions involving rhpn2-a. The following methodology is recommended based on established Xenopus protein interaction studies:

Protocol for rhpn2-a immunoprecipitation:

• Sample preparation:

  • Homogenize Xenopus embryos or tissues in IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitors)

  • Clarify lysates by centrifugation (14,000 × g, 15 min, 4°C)

  • Pre-clear with Protein A/G beads (30 min, 4°C)

• Immunoprecipitation:

  • Option A: For tagged rhpn2-a, use anti-tag antibodies (conjugated to beads)

  • Option B: For endogenous protein, use specific anti-rhpn2-a antibodies

  • Option C: For interactome studies, use purified recombinant rhpn2-a conjugated to beads as bait

• Detection methods:

  • Western blot analysis with appropriate antibodies

  • Mass spectrometry for unbiased identification of binding partners

  • Activity assays to assess functional interactions with Rho GTPases

For identifying transcription factor interactions, techniques successfully applied to other Xenopus proteins, such as electrophoretic mobility shift assays (EMSA), can be adapted for rhpn2-a studies .

How can single-cell RNA-Seq approaches be used to study Rhophilin-2-A expression patterns during Xenopus development and regeneration?

Single-cell RNA sequencing (scRNA-Seq) provides unprecedented resolution for analyzing gene expression patterns at the cellular level. For studying rhpn2-a expression, researchers can adapt established protocols from Xenopus regeneration studies:

Methodology:

• Sample preparation:

  • Collect embryos at different developmental stages or during regeneration processes

  • Dissociate tissues using gentle enzymatic treatment (e.g., 0.25% trypsin-EDTA)

  • Filter through a 40 μm cell strainer to obtain single-cell suspensions

• scRNA-Seq workflow:

  • Use established platforms (10x Genomics, Drop-seq, Smart-seq2)

  • Sequence at a depth of ≥50,000 reads per cell

  • Apply quality control filters (minimum gene count, maximum mitochondrial content)

• Data analysis for rhpn2-a expression:

  • Identify cell populations expressing rhpn2-a

  • Perform differential expression analysis across developmental timepoints

  • Construct pseudotemporal trajectories to track expression changes

  • Identify co-expressed genes for pathway analysis

This approach could reveal whether rhpn2-a is expressed in specific cell populations, such as the recently characterized Regeneration Initiating Cells (RICs) in Xenopus, which comprise up to 10% of basal epidermal cells during regeneration .

What are the optimal experimental conditions for using rhpn2-a in developmental studies?

For developmental studies involving rhpn2-a in Xenopus laevis, researchers should consider the following experimental conditions:

Embryo handling and microinjection:

• Collect and fertilize eggs according to standard protocols

• For microinjection, use calibrated needles (diameter 10-15 μm)

• Inject 250-500 pg of mRNA encoding wild-type or modified rhpn2-a

• Target injections to specific blastomeres based on experimental goals

• Maintain embryos at 18-22°C in 0.1× Marc's Modified Ringer's (MMR) solution

Expression analysis:

• For temporal expression, collect embryos at key developmental stages

• For spatial expression, perform whole-mount in situ hybridization or immunohistochemistry

• For quantitative analysis, use qPCR or western blotting

Functional studies:

• Loss-of-function: morpholino injection (5-20 ng) or CRISPR-Cas9

• Gain-of-function: mRNA injection (250-500 pg)

• Rescue experiments: co-injection of morpholino with modified mRNA

These parameters are derived from successful studies of other Xenopus proteins and can be optimized specifically for rhpn2-a .

How can the purity and activity of Recombinant Xenopus laevis Rhophilin-2-A be verified?

Verifying the purity and activity of recombinant rhpn2-a is crucial for experimental reliability. The following methods are recommended:

Purity assessment:

• SDS-PAGE analysis (expected purity >85%)

• Western blotting with anti-rhpn2-a or anti-tag antibodies

• Mass spectrometry for precise composition analysis

Activity verification:

• GTP-Rho binding assays using purified Rho GTPases

• Pull-down assays with GTPγS-loaded Rho proteins

• Surface plasmon resonance (SPR) for binding kinetics determination

Structural integrity:

• Circular dichroism (CD) spectroscopy for secondary structure analysis

• Thermal shift assays to assess protein stability

• Limited proteolysis to evaluate proper folding

For researchers working with Xenopus embryos, activity can also be assessed through microinjection and functional readouts such as effects on cell morphology or cytoskeletal organization.

What are common pitfalls when working with Recombinant Xenopus laevis proteins and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant Xenopus proteins, including rhpn2-a:

When adapting protocols from mammalian systems to Xenopus proteins, consider species-specific differences in optimal temperature, pH, and buffer composition. For instance, Xenopus proteins often exhibit optimal activity at lower temperatures (18-22°C) compared to mammalian counterparts (37°C).

How can genetic code expansion be combined with FRET imaging to study Rhophilin-2-A interactions in living Xenopus embryos?

Combining genetic code expansion with Förster Resonance Energy Transfer (FRET) imaging provides a powerful approach to study rhpn2-a interactions with spatiotemporal precision in living embryos:

Methodology:

• Construct design:

  • Incorporate an amber codon (UAG) at specific sites in rhpn2-a for UAA insertion

  • Create fusion constructs with appropriate FRET donors/acceptors

  • Design constructs for potential interaction partners (e.g., Rho GTPases)

• Embryo preparation:

  • Co-inject PylRS mRNA (250 pg), PylT (7.5 ng), and rhpn2-a construct mRNA (250 pg) into one-cell stage embryos

  • Add appropriate UAA (e.g., 1 or 5) at 10-50 mM concentration

  • Allow development to desired stage

• FRET imaging:

  • Use confocal microscopy with appropriate filter sets

  • Calculate FRET efficiency through acceptor photobleaching or sensitized emission

  • Apply photocaged UAAs for temporal control of interactions

This approach allows for studying dynamic protein-protein interactions in vivo with subcellular resolution, providing insights into how rhpn2-a interacts with binding partners during specific developmental processes.

What approaches can be used to study the role of Rhophilin-2-A in Xenopus regeneration processes?

Investigating rhpn2-a's role in Xenopus regeneration requires integrating multiple experimental approaches:

• Expression analysis during regeneration:

  • Perform RT-qPCR and in situ hybridization at various timepoints after injury

  • Use single-cell RNA-Seq to identify cell populations expressing rhpn2-a

  • Compare expression between regeneration-competent and -incompetent stages

• Functional perturbation:

  • Deploy CRISPR-Cas9 or morpholinos for loss-of-function studies

  • Use mRNA injection for gain-of-function experiments

  • Apply photoactivatable or small molecule-responsive variants (using UAAs 2, 3, or 5) for temporal control

• Cellular analysis:

  • Examine relationship to Regeneration Initiating Cells (RICs), which comprise up to 10% of basal epidermal cells at 12 hours post-amputation

  • Assess co-expression with RIC markers such as palld.L/S, lep.L, pmepa1.S, and inhba.L

  • Track cellular behaviors using live imaging techniques

• Molecular pathway analysis:

  • Identify rhpn2-a interaction partners during regeneration using immunoprecipitation

  • Assess Rho GTPase activation patterns in wild-type vs. rhpn2-a-perturbed samples

  • Investigate downstream cytoskeletal dynamics using fluorescent reporters

These approaches can reveal whether rhpn2-a contributes to the remarkable regenerative capabilities of Xenopus and identify the molecular mechanisms involved.

What are emerging technologies that may advance Rhophilin-2-A research in Xenopus models?

Several cutting-edge technologies hold promise for advancing rhpn2-a research in Xenopus models:

• Spatial transcriptomics and proteomics:

  • Combining single-cell resolution with spatial information

  • Mapping rhpn2-a expression patterns within intact tissues

  • Correlating with cellular behaviors during development and regeneration

• Advanced genetic code expansion applications:

  • Incorporation of multiple distinct UAAs into a single protein

  • Engineering photoswitchable or chemically responsive rhpn2-a variants

  • Creating sensors for detecting rhpn2-a activity in vivo

• Genome editing technologies:

  • CRISPR-Cas9 base editors for precise modification of rhpn2-a

  • Inducible or tissue-specific knockout systems

  • Knockin of fluorescent tags at endogenous loci

• Microfluidics and organ-on-chip technologies:

  • Creating simplified models of Xenopus tissues

  • High-throughput screening of rhpn2-a modulators

  • Real-time analysis of cellular dynamics

These technologies promise to provide deeper insights into rhpn2-a function and regulation, potentially revealing novel roles in development, regeneration, and disease processes.

How can computational approaches enhance our understanding of Rhophilin-2-A function in Xenopus laevis?

Computational approaches offer powerful complementary methods for investigating rhpn2-a function:

• Structural prediction and analysis:

  • Homology modeling based on related proteins

  • Molecular dynamics simulations to predict protein flexibility

  • Docking studies to identify potential binding partners

• Evolutionary analysis:

  • Comparison of rhpn2-a across species to identify conserved domains

  • Examination of selection pressures on functional domains

  • Reconstruction of evolutionary relationships with other Rho effectors

• Network biology approaches:

  • Construction of protein-protein interaction networks

  • Pathway enrichment analysis from expression data

  • Integration of multi-omics data to predict functional roles

• Machine learning applications:

  • Prediction of post-translational modifications

  • Classification of expression patterns across developmental stages

  • Identification of novel regulatory elements in the rhpn2-a promoter