Recombinant Xenopus laevis Coiled-coil domain-containing protein 47 (ccdc47)

What is CCDC47 and what role does it play in Xenopus laevis development?

CCDC47, also known as calumin, is a Ca²⁺-binding ER transmembrane protein involved in embryogenesis and development. In vertebrates, including Xenopus laevis, CCDC47 appears to be essential for early development. The importance of this protein is underscored by studies in mouse models, where loss of Ccdc47 leads to embryonic lethality with observed delayed development, atrophic neural tubes, heart abnormalities, and a paucity of blood cells in the dorsal aorta .

CCDC47 contains a globular N-terminal domain and a long, flexible C-terminal coiled-coil structure that extends to the nascent chain at the ribosome exit tunnel, suggesting its importance in protein synthesis during development . Truncating this conserved motif causes developmental disorders in humans, highlighting its functional significance . In Xenopus laevis specifically, while less extensively studied than in mammals, CCDC47 likely contributes to proper neural development and calcium homeostasis during embryogenesis, which is critical for neural tube formation and early developmental processes.

How does CCDC47 function as a Ca²⁺-binding protein in the endoplasmic reticulum?

CCDC47 functions as a calcium-binding protein in the endoplasmic reticulum with low affinity but high capacity for calcium ions . To study this function in Xenopus laevis, researchers can employ several methodological approaches:

• Calcium imaging using fluorescent indicators in Xenopus cells with normal or altered CCDC47 expression can reveal changes in intracellular calcium dynamics.

• Store-operated calcium entry (SOCE) assays can be conducted by depleting ER calcium stores with thapsigargin and measuring subsequent calcium influx.

• IP₃-mediated calcium release can be assessed by stimulating cells with agonists that generate IP₃.

• Patch-clamp electrophysiology can directly measure calcium currents.

Experimental evidence from mouse embryonic fibroblasts (MEFs) with Ccdc47 knockout showed impaired Ca²⁺ signaling , suggesting that CCDC47 is critical for normal calcium homeostasis. In human studies, cells from individuals with predicted damaging CCDC47 alleles demonstrated decreased total ER Ca²⁺ storage, impaired Ca²⁺ signaling mediated by the IP₃R Ca²⁺ release channel, and reduced ER Ca²⁺ refilling via store-operated Ca²⁺ entry . Similar approaches in Xenopus laevis would likely yield valuable insights into CCDC47's role in calcium regulation during amphibian development.

What are the structural features of CCDC47 in Xenopus laevis?

CCDC47 in Xenopus laevis, similar to its mammalian counterparts, possesses a distinctive structure characterized by:

• A globular N-terminal domain anchored in the ER membrane

• A long, extended C-terminal coiled-coil domain that curls out of the membrane

Structural analyses using cryo-electron microscopy have revealed that the coiled-coil domain extends along the ribosome surface and terminates in a long helical extension that reaches the nascent chain at the ribosome exit tunnel . Structure prediction tools like RaptorX-Contact have been used to generate models of this region, showing that the globular domain of CCDC47 contacts eL6 and rRNA H25, while the conserved and positively charged coiled-coil wedges between Sec61 and rRNA H24 .

The entire translocon complex, including CCDC47, extends approximately 90 x 120 x 140 Å, with significant mass on both sides of the membrane . This structural arrangement positions the cytosolic domains of CCDC47 near the nascent chain as it emerges from the ribosome exit tunnel, suggesting a role in protein biogenesis .

Why is Xenopus laevis an ideal model organism for studying CCDC47?

Xenopus laevis offers several significant advantages for studying CCDC47 function:

• Well-characterized developmental stages allow precise temporal analysis of CCDC47 expression and function .

• The Xenopus genome has been sequenced, facilitating genetic manipulation and analysis .

• Rapid external development and large embryo size enable easy observation and manipulation .

• Xenopus cell cultures are ideal for long periods of live imaging because they are easily obtained and maintained without requiring special culture conditions .

• Xenopus neural tube and retinal explants can be cultured to study CCDC47's role in neuronal development .

• Xenopus laevis has relatively large growth cones (10-30 μm in diameter) compared to other vertebrates, making cytoskeletal and protein imaging much easier .

These advantages make Xenopus laevis particularly suitable for investigating calcium signaling pathways involving CCDC47, as the larger cell size facilitates calcium imaging and the well-established developmental timeline allows for precise correlation between CCDC47 function and specific developmental events.

What phenotypes are associated with CCDC47 dysfunction in vertebrates?

CCDC47 dysfunction is associated with several distinct phenotypes across vertebrate species:

• In humans, bi-allelic variants in CCDC47 cause a complex multisystem disorder characterized by:

  • Woolly hair

  • Liver dysfunction

  • Pruritus

  • Dysmorphic features

  • Hypotonia

  • Global developmental delay

• In mouse models, complete loss of Ccdc47 leads to:

  • Embryonic lethality

  • Delayed development

  • Atrophic neural tubes

  • Heart abnormalities

  • Reduced blood cells in the dorsal aorta

• At the cellular level, cells with CCDC47 dysfunction exhibit:

  • Decreased total ER Ca²⁺ storage

  • Impaired Ca²⁺ signaling mediated by the IP₃R Ca²⁺ release channel

  • Reduced ER Ca²⁺ refilling via store-operated Ca²⁺ entry

These findings suggest that CCDC47 plays essential roles in calcium homeostasis and protein biogenesis during development. The specific phenotypes in Xenopus laevis with CCDC47 dysfunction have not been extensively documented in the provided search results, but based on conservation of function across vertebrates, similar developmental abnormalities would be expected.

What experimental approaches can be used to express and purify recombinant Xenopus laevis CCDC47?

Expressing and purifying recombinant Xenopus laevis CCDC47 requires specialized approaches due to its transmembrane nature:

Expression systems:

• Bacterial expression (E. coli BL21(DE3) or C41/C43 strains optimized for membrane proteins)

• Insect cell expression (Sf9 or High Five cells using baculovirus)

• Mammalian cell expression (HEK293 or CHO cells)

Expression optimization strategies:

• Use different affinity tags (His6, GST, or MBP) at either terminus

• Lower expression temperature (16-18°C) to improve folding

• Supplement media with calcium to stabilize the protein

• Test different induction protocols

Purification protocol:

• Cell lysis in detergent-containing buffer (DDM, LMNG, or GDN)

• Initial capture using affinity chromatography

• Ion exchange chromatography for further purification

• Size exclusion chromatography for final polishing

Quality assessment:

• SDS-PAGE and Western blotting

• Circular dichroism to assess secondary structure

• Calcium binding assays to confirm functionality

• Limited proteolysis to identify stable domains

For structural studies of CCDC47, researchers have used RaptorX-Contact to generate models of the large cytosolic region, revealing a long and flexible C-terminal coiled-coil extending from a globular N-terminal domain . This computational approach complemented experimental structural determination and can guide the design of expression constructs for different functional domains of the protein.

How can researchers visualize CCDC47 localization and dynamics in Xenopus laevis growth cones?

Visualizing CCDC47 in Xenopus laevis growth cones leverages the unique advantages of this model system, particularly the large growth cone size (10-30 μm in diameter) :

Fluorescent protein tagging approaches:

• Express fluorescently tagged CCDC47 (GFP-CCDC47 or CCDC47-mCherry) in Xenopus neurons

• Culture neurons from neural tube explants following established Xenopus protocols

• Co-express with ER markers (e.g., Sec61β-GFP) to confirm localization

Microscopy techniques:

• Live-cell confocal microscopy for time-lapse imaging of CCDC47 dynamics

• Super-resolution microscopy (SIM, STED, or SMLM) for precise subcellular localization

• FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility

For studying endogenous CCDC47:

• Immunofluorescence using specific antibodies against CCDC47

• Optimize fixation protocols to preserve growth cone morphology

• Use tyramide signal amplification for enhanced detection sensitivity

Correlative approaches:

• Combine CCDC47 imaging with calcium indicators to link localization with calcium dynamics

• Perform simultaneous DIC imaging to correlate CCDC47 dynamics with growth cone behavior

• Use automated tracking software to quantify CCDC47 movements during growth cone advance

The relatively large size of Xenopus growth cones provides excellent imaging capabilities compared to other vertebrate models, making it possible to distinguish localization patterns within different growth cone regions (peripheral domain, transitional zone, and central domain) .

What are the best methods to study CCDC47's role in Ca²⁺ signaling in Xenopus laevis neural development?

Studying CCDC47's role in Ca²⁺ signaling during Xenopus laevis neural development requires combining molecular manipulation with calcium imaging techniques:

CCDC47 manipulation approaches:

• Antisense morpholinos targeting CCDC47 mRNA

• CRISPR-Cas9 genome editing of CCDC47

• Overexpression of wild-type or mutant CCDC47

• Expression of disease-associated CCDC47 variants identified in human patients

Calcium imaging methods:

• Genetically encoded calcium indicators (GCaMP6f) for cytosolic calcium

• ER-targeted calcium indicators (G-CEPIA1er) for direct monitoring of ER calcium

• Dual-wavelength ratiometric imaging for quantitative calcium measurements

• High-speed spinning disk confocal microscopy for capturing rapid calcium transients

Experimental paradigms:

• Measure store-operated calcium entry following thapsigargin-induced store depletion

• Assess IP₃-mediated calcium release using receptor agonists

• Monitor spontaneous calcium oscillations during neural development

• Evaluate calcium dynamics during growth cone guidance decisions

Correlative analyses:

• Link calcium signaling patterns to morphological outcomes

• Perform calcium imaging in different CCDC47 mutant backgrounds

• Conduct rescue experiments with wild-type CCDC47 following knockdown

These approaches would build upon findings that human cells with CCDC47 variants show impaired Ca²⁺ signaling mediated by the IP₃R Ca²⁺ release channel and reduced ER Ca²⁺ refilling via store-operated Ca²⁺ entry , extending these observations to the developmental context of Xenopus laevis.

How does CCDC47 interact with the translocon complex during multi-pass membrane protein biogenesis in Xenopus laevis?

Investigating CCDC47's interaction with the translocon complex requires sophisticated biochemical and imaging approaches:

Structural insights:

• Cryo-electron microscopy has revealed that CCDC47 is part of a novel human translocon complex involved in multi-pass membrane protein biogenesis

• The globular domain of CCDC47 contacts eL6 and rRNA H25, while its coiled-coil extends between Sec61 and rRNA H24

• The C-terminal coiled-coil of CCDC47 extends to the nascent chain at the ribosome exit tunnel

Interaction analysis methods:

• Co-immunoprecipitation of CCDC47 with translocon components (Sec61α, TMCO1, TMEM147, Nicalin)

• Crosslinking mass spectrometry to identify specific interaction sites

• FRET microscopy between fluorescently tagged CCDC47 and translocon proteins

Functional assays:

• In vitro translation assays using Xenopus egg extracts with manipulated CCDC47 levels

• Analysis of membrane protein integration efficiency in CCDC47-depleted systems

• Assessment of translational pausing during membrane protein synthesis

Imaging approaches:

• Multi-color super-resolution microscopy of CCDC47 and translocon components

• Live imaging of fluorescently tagged CCDC47 during active translation

The translocon complex extends approximately 90 x 120 x 140 Å, with CCDC47 positioned to interact with the nascent chain as it emerges from the ribosome . This strategic positioning suggests a critical role in multi-pass membrane protein biogenesis, potentially serving as a molecular chaperone during the complex folding process of transmembrane domains.

What are the most effective techniques to investigate CCDC47 binding partners in Xenopus laevis?

Identifying CCDC47 binding partners in Xenopus laevis requires a comprehensive protein-protein interaction analysis approach:

Affinity-based methods:

• Immunoprecipitation of endogenous CCDC47 followed by mass spectrometry

• Tandem affinity purification using tagged CCDC47 expressed in Xenopus embryos

• GST pull-down assays with recombinant CCDC47 domains

Proximity-based approaches:

• BioID fusion proteins to biotinylate proximal proteins in living cells

• APEX2 proximity labeling for rapid identification of neighboring proteins

• Proximity ligation assay (PLA) to visualize protein interactions in situ

Validation techniques:

• Co-immunoprecipitation and Western blotting for specific candidate interactors

• FRET or BiFC assays in Xenopus cells to confirm direct interactions

• Co-localization studies using confocal microscopy

Context-specific analyses:

• Perform interaction studies under calcium-depleted and calcium-replete conditions

• Investigate developmental stage-specific interaction partners

• Compare binding partners in normal versus calcium signaling-perturbed contexts

Known interaction partners of CCDC47 include components of the translocon complex such as Sec61α, TMCO1, TMEM147, and Nicalin . These interactions have been confirmed through cryo-electron microscopy and crosslinking studies, revealing that CCDC47 satisfies multiple intra- and inter-molecular cross-links to Sec61α, to the flexible N-terminus of Sec61β, to uL22, eL31 and eL32, and to the TMCO1 coiled-coil and C-terminal helix .

How can researchers generate and validate CCDC47 knockdown or knockout models in Xenopus laevis?

Generating and validating CCDC47 loss-of-function models in Xenopus laevis requires several complementary approaches:

Knockdown strategies:

• Design morpholino antisense oligonucleotides targeting either:

  • Translation start site of CCDC47 mRNA

  • Splice junctions to disrupt CCDC47 mRNA processing

• Microinject morpholinos into fertilized Xenopus eggs at the 1-2 cell stage

Knockout approaches:

• CRISPR-Cas9 genome editing with sgRNAs targeting conserved regions of CCDC47

• Design sgRNAs targeting both homeologs (L and S versions) of the gene due to Xenopus laevis' pseudo-tetraploid nature

• Deliver Cas9 protein complexed with in vitro transcribed sgRNAs via microinjection

Validation methods:

• Molecular validation:

  • RT-qPCR and Western blotting to confirm reduced CCDC47 expression

  • T7 endonuclease I assay or direct sequencing to confirm genomic modifications

• Functional validation:

  • Calcium imaging to assess ER calcium handling

  • ER stress marker analysis (BiP, CHOP)

  • Evaluation of protein synthesis and folding

Rescue experiments:

• Co-inject wild-type CCDC47 mRNA with knockdown/knockout reagents

• Rescue with human CCDC47 to test conservation of function

• Structure-function analysis using truncated or mutated CCDC47 variants

The importance of proper validation is underscored by findings in mouse models, where complete loss of Ccdc47 leads to embryonic lethality , suggesting that complete knockout in Xenopus may also produce severe developmental phenotypes that require careful characterization.

What bioinformatic approaches are useful for comparative analysis of CCDC47 across species, including Xenopus laevis?

Comparative analysis of CCDC47 across species requires sophisticated bioinformatic approaches:

Sequence analysis:

• Multiple sequence alignment of CCDC47 orthologs from diverse vertebrates

• Conservation analysis of functional domains and motifs

• Identification of species-specific variations in the coiled-coil region

Structural prediction:

• Generate 3D models using tools like RaptorX-Contact

• Compare predicted structures across species

• Identify conserved structural elements, particularly in the coiled-coil domain

Evolutionary analysis:

• Phylogenetic reconstruction of CCDC47 evolution

• Calculation of selection pressures on different protein domains

• Comparison between L and S homeologs in Xenopus laevis

Functional prediction:

• Analysis of conserved binding sites for calcium and protein partners

• Prediction of post-translational modification sites

• Identification of conserved gene regulatory elements

Expression analysis:

• Compare tissue-specific expression patterns across species

• Analyze developmental expression timing in different vertebrates

• Identify co-expressed genes as potential functional partners

These approaches would build upon existing structural data, such as the identification of CCDC47's globular N-terminal domain and long, flexible C-terminal coiled-coil that extends from the membrane and terminates at the ribosome exit tunnel , providing evolutionary context for these functionally important features.

How do mutations in CCDC47 affect calcium homeostasis in Xenopus laevis embryonic development?

Investigating how CCDC47 mutations affect calcium homeostasis in Xenopus laevis embryonic development requires integrated approaches:

Mutation strategies:

• Introduce disease-associated CCDC47 mutations identified in human patients

• Create truncations of the C-terminal coiled-coil domain that has been shown to be functionally important

• Mutate predicted calcium-binding sites based on structural analysis

Calcium homeostasis assessment:

• Measure ER calcium levels using ER-targeted calcium indicators

• Assess store-operated calcium entry following store depletion

• Evaluate IP₃-mediated calcium release

• Monitor spontaneous calcium oscillations during development

Developmental phenotyping:

Molecular consequences:

• Measure ER stress markers (BiP, CHOP)

• Evaluate unfolded protein response activation

• Assess membrane protein synthesis and trafficking

Human studies have shown that cells from individuals with bi-allelic CCDC47 variants exhibit decreased total ER Ca²⁺ storage, impaired Ca²⁺ signaling mediated by the IP₃R Ca²⁺ release channel, and reduced ER Ca²⁺ refilling via store-operated Ca²⁺ entry . Similar defects would likely occur in Xenopus embryos with CCDC47 mutations, potentially leading to developmental abnormalities in calcium-dependent processes such as neural tube formation, neuronal differentiation, and heart development.