Recombinant Macaca fascicularis Transmembrane protein C6orf70 homolog (QtsA-18153)

What is the C6orf70 protein and what is its known function in Macaca fascicularis?

C6orf70 is predicted to code for a potential multi-pass membrane protein with currently undefined specific functions. In human cell lines, C6orf70 demonstrates primarily a cytoplasmic vesicular puncta-like distribution pattern . The Macaca fascicularis homolog shares significant sequence similarity with the human version, reflecting the close evolutionary relationship between these species . Studies of C6orf70 in both human and animal models have demonstrated its critical role in neuronal migration during brain development . The protein's importance has been established through knockdown experiments that resulted in periventricular nodular heterotopia (PNH), a condition where neurons fail to migrate properly .

How does the genomic context of C6orf70 differ between humans and Macaca fascicularis?

What experimental evidence confirms the role of C6orf70 in neuronal migration disorders?

In vivo silencing experiments provide compelling evidence for C6orf70's role in neuronal migration. When C6orf70 was silenced using shRNA constructs in the developing rat neocortex, researchers observed a striking arrest of neuronal migration, resulting in periventricular heterotopia . The specificity of this effect was confirmed by rescue experiments: concomitant expression of wild-type human C6orf70 protein alongside the silencing construct successfully prevented the migration defects . This establishes a direct causal relationship between C6orf70 function and proper neuronal migration.

Comparative experiments silencing the adjacent genes Phf10 and Dll1 resulted only in slightly delayed migration but not periventricular nodular heterotopia, demonstrating the specific importance of C6orf70 in this process . Additional evidence comes from clinical studies where patients with developmental brain abnormalities harboring a 1.2 Mb deletion in chromosome 6q27 exhibited periventricular nodular heterotopia alongside other neurological features .

How do mutations in C6orf70 affect protein stability and subcellular distribution?

These findings suggest that proper subcellular targeting of C6orf70 is essential for its function in neuronal migration. The vesicular distribution pattern implies a potential role in intracellular trafficking processes that may be critical for neuronal migration. Further investigation using live-cell imaging techniques would provide additional insights into the dynamic behavior of wild-type versus mutant C6orf70 proteins.

What approaches can be used to characterize protein-protein interactions of C6orf70?

Characterizing the interactome of C6orf70 is essential for understanding its functional mechanisms. Several complementary approaches are recommended:

• Co-immunoprecipitation (Co-IP) combined with mass spectrometry to identify binding partners

• Proximity labeling techniques (BioID or APEX) to map the protein's neighborhood within living cells

• Yeast two-hybrid screening to identify direct protein interactions

• Fluorescence resonance energy transfer (FRET) to validate specific interactions in living cells

The vesicular distribution pattern of C6orf70 suggests potential interactions with membrane trafficking machinery . Candidate interaction partners might include components of the cytoskeleton, vesicular transport proteins, or membrane-associated signaling complexes involved in neuronal migration.

What expression systems are optimal for producing recombinant Macaca fascicularis C6orf70 homolog?

Based on the membrane protein characteristics of C6orf70, the following expression systems should be considered for recombinant production:

What purification strategies are most effective for recombinant C6orf70?

As a predicted multi-pass membrane protein, C6orf70 presents significant challenges for purification. A systematic approach is recommended:

• Detergent screening - Test a panel of detergents (DDM, LMNG, digitonin) for optimal solubilization

• Affinity purification - Utilize epitope tags (His, FLAG, or Strep-tag II) positioned to minimize interference with protein function

• Size exclusion chromatography - For further purification and to assess protein homogeneity

• Reconstitution - Consider nanodiscs or liposomes for functional studies requiring membrane environment

Protein stability should be monitored throughout the purification process using techniques such as differential scanning fluorimetry to identify optimal buffer conditions.

What techniques are most effective for studying C6orf70 function in neuronal migration?

Multiple complementary approaches provide insights into C6orf70's role in neuronal migration:

• In utero electroporation - For targeted gene manipulation in developing brain, as demonstrated by successful C6orf70 silencing experiments in rat neocortex

• Ex vivo brain slice cultures - To monitor neuronal migration in a controlled environment

• Time-lapse imaging - To visualize migration dynamics in real-time

• CRISPR/Cas9 genome editing - To generate precise mutations mirroring those found in patients

The successful rescue experiments reported in the literature provide a powerful validation model: silencing endogenous C6orf70 followed by expressing wild-type or mutant variants allows direct assessment of functional consequences .

How can researchers investigate the molecular mechanisms of C6orf70 in cellular models?

Understanding the molecular mechanisms requires integrating multiple experimental approaches:

• Subcellular fractionation combined with western blotting to determine precise localization

• Live-cell imaging with fluorescently tagged C6orf70 to track dynamics

• Proteomic analysis of vesicles containing C6orf70 to identify associated proteins

• Functional readouts such as vesicular trafficking assays or migration assays

Particular attention should be paid to the vesicular puncta-like distribution pattern, which suggests a potential role in intracellular trafficking processes . Investigating how this distribution changes during neuronal migration would provide valuable mechanistic insights.

How does the C6orf70 homolog in Macaca fascicularis compare with other primate species?

The whole-genome sequencing of Macaca fascicularis has enabled comparative genomic analysis of protein-coding genes between macaque species and humans . For membrane proteins like C6orf70, sequence conservation can vary across different functional domains. The table below summarizes comparative data:

This high conservation, particularly in transmembrane domains, suggests strong evolutionary pressure to maintain the protein's structure and function, supporting its critical role in neuronal development .

What can disease models tell us about C6orf70 function across species?

The periventricular nodular heterotopia phenotype observed from C6orf70 dysfunction provides a valuable disease model. In humans, mutations or haploinsufficiency of C6orf70 within the 6q27 deletion syndrome are associated with periventricular nodular heterotopia, corpus callosum dysgenesis, colpocephaly, cerebellar hypoplasia, and polymicrogyria . These anatomical abnormalities typically manifest with epilepsy, ataxia, and cognitive impairment.

Knockdown experiments in rat models produced comparable periventricular heterotopia, suggesting conservation of function across species . The observed rescue of migration defects by wild-type human C6orf70 in rat models provides strong evidence for functional conservation . These findings support the utility of Macaca fascicularis as an appropriate model for studying human neurological disorders associated with C6orf70 dysfunction.

What controls are essential when studying recombinant Macaca fascicularis C6orf70?

Robust experimental design requires several critical controls:

• Expression vector controls - Empty vector and irrelevant protein expressions to control for overexpression artifacts

• Species-specific controls - Comparing recombinant proteins from human and Macaca fascicularis to identify species-specific differences

• Subcellular localization controls - Markers for various cellular compartments to accurately determine protein localization

• Functional rescue controls - Using both wild-type and mutant versions in rescue experiments to demonstrate specificity

These controls are particularly important when evaluating the subcellular distribution patterns of C6orf70, which shows primarily a cytoplasmic vesicular puncta-like distribution in human cell lines .

How should researchers address potential experimental artifacts when studying membrane proteins like C6orf70?

Membrane proteins present unique challenges that require specific considerations:

• Overexpression artifacts - Titrate expression levels; compare stable versus transient expression

• Tag interference - Test multiple tag positions and types; validate with untagged protein where possible

• Fixation artifacts - Compare multiple fixation protocols for immunohistochemistry

• Detergent effects - Screen detergents for minimal disruption of protein-protein interactions

• Species-specific differences - Use cells derived from relevant species when possible

When studying C6orf70's vesicular puncta-like distribution, these considerations are particularly important as membrane protein localization can be easily disrupted by experimental manipulations .

How can findings from Macaca fascicularis C6orf70 studies inform human neurological disorder research?

The high genomic similarity between Macaca fascicularis and humans (approximately 93-95%) makes findings from macaque studies particularly relevant to human neurological disorders . For C6orf70 specifically, the following translational pathways exist:

• Biomarker development - Identifying measurable changes associated with C6orf70 dysfunction

• Target validation - Confirming C6orf70's role in disease pathways before therapeutic development

• Phenotypic screening - Using C6orf70 function as a readout to identify compounds that restore normal function

• Safety assessment - Leveraging Macaca fascicularis models to evaluate potential therapeutics

The established role of C6orf70 in periventricular heterotopia provides a clear link to human neurological disorders, particularly those involving neuronal migration defects and associated epilepsy .

What imaging techniques are most informative for assessing C6orf70-related phenotypes?

Based on the known association of C6orf70 with neuronal migration disorders and periventricular heterotopia, several imaging approaches are particularly valuable:

• Magnetic Resonance Imaging (MRI) - For structural assessment of periventricular heterotopia, corpus callosum dysgenesis, and other neuroanatomical features associated with C6orf70 dysfunction

• Diffusion Tensor Imaging (DTI) - To evaluate white matter tract integrity, which may be affected in C6orf70-related disorders

• Functional MRI (fMRI) - To assess potential alterations in brain activity and connectivity

• Super-resolution microscopy - For detailed analysis of C6orf70's subcellular localization in experimental models

These techniques complement each other by providing information at different scales, from whole-brain structure to subcellular protein distribution, creating a comprehensive picture of C6orf70's role in brain development and function.