Recombinant Xenopus laevis Daple-like protein (ccdc88c), partial

What are the optimal storage conditions for Recombinant Xenopus laevis Daple-like protein?

The recombinant protein should be stored at -20°C, and for extended storage, conserved at -20°C or -80°C . Repeated freezing and thawing should be avoided to maintain protein integrity. Working aliquots can be safely stored at 4°C for up to one week . For reconstitution, we recommend briefly centrifuging the vial before opening and reconstituting in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol (final concentration) for long-term storage .

What structural domains characterize Xenopus Daple-like protein and how do they compare to mammalian homologs?

Xenopus Daple-like protein (xDal) contains several functional domains comparable to its mammalian counterparts:

The protein is evolutionarily conserved, with the Xenopus ortholog showing functional similarities in Wnt pathway modulation .

What expression patterns does Daple-like protein exhibit during Xenopus development?

Daple exhibits a specific temporal expression pattern during Xenopus development:

RNA-seq data reveals that xMPDZ mRNA expression closely resembles the temporal pattern of Xenopus DAPLE expression during embryonic development .

How should researchers design experiments to study the GBA motif function of Daple in Xenopus?

When investigating the GBA motif function in Xenopus Daple-like protein, researchers should:

• Design mRNA constructs with wild-type and mutant forms of the GBA motif for microinjection into Xenopus embryos

• Utilize GBAi (a specific inhibitor of GPCR-independent G protein signaling) to block GBA-mediated processes in vivo

• Employ proper controls including uninjected embryos and embryos injected with control mRNAs

• Monitor gastrulation movements and convergent extension defects as phenotypic readouts

• Combine with biochemical assays to measure downstream G protein activation

• Consider co-injection experiments with other pathway components to assess specificity

The GBA motif's role can be assessed by comparing phenotypes between embryos injected with wild-type DAPLE, GBA-mutant DAPLE, and co-injection with GBAi inhibitor .

What methodological approaches can determine the interaction between Daple and Dishevelled in Xenopus models?

To characterize the Daple-Dishevelled interaction in Xenopus:

• Perform co-immunoprecipitation from embryo lysates at different developmental stages

• Use yeast two-hybrid assays with deletion constructs to map interaction domains

• Employ fluorescently tagged proteins for co-localization studies in Xenopus cells

• Conduct FRET analysis in live embryos to visualize the dynamics of interaction

• Implement domain-specific mutagenesis to identify critical residues for binding

• Assess functional consequences through phenotypic analysis of embryos expressing interaction-deficient mutants

These approaches allow comprehensive characterization of both physical interaction and functional significance in a developmental context .

How does Xenopus Daple-like protein modulate Wnt signaling pathways during development?

Xenopus Daple-like protein (xDal) functions as a modulator of both canonical and noncanonical Wnt signaling pathways:

Mechanistically, xDal interacts directly with Dishevelled, a core component of Wnt signaling . Additionally, through its GBA motif, xDal activates heterotrimeric G-proteins, which further reinforces noncanonical Wnt signaling via Rho-dependent actomyosin contractility .

What is the relationship between Daple and the JNK pathway in normal development versus pathological conditions?

The relationship between Daple and JNK pathway activation reveals a dual role:

How can Xenopus models of Daple dysfunction inform our understanding of human neurodevelopmental disorders?

Xenopus models provide valuable insights into human neurodevelopmental disorders associated with Daple dysfunction:

• Both CCDC88C (encoding DAPLE) and MPDZ are genetically linked to nonsyndromic congenital hydrocephalus (NSCH) in humans

• Dorsal injection of DAPLE mRNA into Xenopus embryos causes gastrulation defects, mimicking early developmental abnormalities

• Loss of DAPLE in Xenopus impairs apical constriction of neuroepithelial cells during neurulation, potentially explaining neural tube defects

• The JNK pathway hyperactivation observed in mutant CCDC88C can be studied in Xenopus to understand mechanisms of neurodegeneration

• Xenopus allows investigation of both loss-of-function and gain-of-function mutations in a vertebrate context

The similar expression patterns of DAPLE and MPDZ during Xenopus development support their cooperative role in brain ventricle formation, providing a model system to study hydrocephalus pathogenesis .

What are the implications of the p.R464H mutation found in spinocerebellar ataxia for protein function and experimental design?

The p.R464H mutation in CCDC88C has significant implications:

The mutation occurs in the evolutionarily conserved HOOK domain of CCDC88C, suggesting its functional importance . Experiments should compare wild-type and mutant proteins, using JNK inhibitors like SP600125 to validate pathway specificity . The gain-of-function nature of this mutation highlights the importance of precise regulation of DAPLE activity in normal physiology.

What cutting-edge biochemical approaches can distinguish between different functional domains of Xenopus Daple-like protein?

Advanced biochemical approaches to dissect Daple functional domains include:

• CRISPR/Cas9-mediated genome editing in Xenopus to create domain-specific knockouts

• Proximity labeling (BioID/TurboID) to identify domain-specific interactomes in different developmental contexts

• Single-molecule FRET to detect conformational changes upon binding to different partners

• Hydrogen-deuterium exchange mass spectrometry to map domain interactions and structural dynamics

• Domain-swapping experiments between Xenopus and human DAPLE to identify species-specific functions

• Optogenetic tools to achieve spatiotemporal control of specific domain activities during development

These approaches allow precise manipulation and analysis of individual domains while maintaining the contextual information of the full protein and developmental system.

How can researchers differentiate between GPCR-dependent and GPCR-independent G protein signaling mediated by Daple?

To distinguish between these signaling modes:

• Utilize GBAi, a specific peptide inhibitor that blocks the GBA motif-mediated G protein activation without affecting GPCR signaling

• Compare phenotypes in Xenopus embryos treated with GBAi versus broad-spectrum G protein inhibitors

• Generate GBA motif mutants that specifically disrupt G protein binding without affecting other functions

• Employ BRET-based biosensors to monitor G protein activation kinetics with temporal precision

• Conduct experiments in the presence of selective GPCR antagonists to block receptor-mediated signaling

• Perform rescue experiments in GPCR-deficient backgrounds with wild-type and mutant DAPLE

These approaches have successfully revealed that the GBA motif of DAPLE is specifically required for its effects on gastrulation and convergent extension in Xenopus embryos, distinguishing this activity from classical GPCR signaling .

How does the interaction between DAPLE and MPDZ contribute to neural development?

DAPLE and MPDZ interact to regulate neural development through:

• Direct binding via DAPLE's PDZ-binding motif (PBM) and MPDZ's PDZ domains

• Cooperative promotion of apical constriction in neuroepithelial cells

• Combined regulation of cell junction formation during neural tube closure

• Co-expression during critical periods of neural development in Xenopus

This interaction is particularly significant as mutations in both genes are linked to nonsyndromic congenital hydrocephalus in humans . The cooperative function suggests that disruption of either protein can lead to similar developmental defects, explaining their genetic association with the same clinical condition.

What experimental strategies can identify novel interaction partners of Xenopus Daple-like protein?

To identify novel interaction partners:

• Perform immunoprecipitation coupled with mass spectrometry at different developmental stages

• Utilize yeast two-hybrid screens with different functional domains as baits

• Implement BioID or APEX2 proximity labeling in Xenopus embryos to capture transient interactions

• Conduct cross-linking mass spectrometry to identify direct binding partners

• Perform co-fractionation analysis to identify proteins in the same complexes

• Use protein arrays to screen for direct interactions with candidate partners

These approaches should be performed at different developmental stages to capture dynamic interaction networks that may change during gastrulation, neurulation, and later developmental processes.