CCDC51 Antibody

What is CCDC51 and where is it primarily localized in cells?

CCDC51, also called MITOK, is a 411-amino acid mitochondrial protein with a predicted N-terminal mitochondrial targeting sequence, a coiled-coil domain (amino acids 111-173), and two transmembrane helical domains (TM1: amino acids 202-222 and TM2: amino acids 387-407). It functions as a mitochondrial inner membrane protein with both N- and C-termini exposed toward the internal matrix, while the region between the two transmembrane domains (amino acids 223-386) resides in the intermembrane space . Immunolocalization studies have confirmed CCDC51/MITOK's presence in mitochondria, where it partially colocalizes with mitochondrial markers like ATP synthase subunit beta. Interestingly, CCDC51 also shows nuclear localization in some cell types, as previously observed in U-2 OS cells .

What methods are most effective for detecting endogenous CCDC51 in tissue samples?

For effective detection of endogenous CCDC51 in tissue samples, immunofluorescence staining using specific anti-CCDC51 antibodies combined with mitochondrial markers (such as anti-ATP synthase antibodies or MitoTracker probes) provides reliable results. This approach has successfully demonstrated CCDC51 expression in human fibroblasts, HeLa cells, and various retinal tissues . For retinal tissues specifically, both fluorescent secondary antibodies and immunohistochemical methods using horseradish peroxidase have proven effective. When examining retinal sections, CCDC51 shows distinct localization in the inner segments of both rod and cone photoreceptors, which aligns with the high concentration of mitochondria in these cellular compartments .

How can I verify CCDC51 antibody specificity for my experimental applications?

To verify CCDC51 antibody specificity, a multi-faceted approach is recommended. First, compare staining patterns with established mitochondrial markers like ATP synthase subunit beta or MitoTracker in wild-type cells. Second, implement CCDC51 knockdown controls using siRNA (such as the validated sequences: 5'-AGACTTGGTGGGACAGATATT-3' or 5'-GACTCAACGAGGTTCGAGATT-3') to confirm reduction or elimination of the signal . Third, perform western blot analysis to verify the antibody detects a protein of the expected size (approximately 45 kDa). Finally, consider using GFP-tagged CCDC51 (with GFP inserted after the mitochondrial targeting sequence) as a positive control to compare localization patterns with the antibody staining of endogenous protein .

How should I design experiments to study CCDC51's role in mitochondrial dynamics?

When investigating CCDC51's role in mitochondrial dynamics, a comprehensive approach including both loss-of-function and gain-of-function experiments is recommended. For loss-of-function studies, both transient siRNA knockdown and stable CRISPRi approaches have proven effective . Importantly, these approaches reveal different phenotypes based on duration and extent of protein depletion: acute knockdown (72 hours) primarily causes mitochondrial hyperfusion, while prolonged depletion (120 hours) leads to the formation of distinctive lamellar mitochondrial structures .

For gain-of-function studies, overexpression of GFP-tagged CCDC51 (with GFP inserted after the mitochondrial targeting sequence) can promote Drp1-dependent mitochondrial fission. To examine dynamic changes, challenge cells with agents that induce mitochondrial fission (such as BAPTA-AM) and monitor morphological changes at different time points (10 and 30 minutes) through confocal microscopy . Always include appropriate controls, such as Drp1 knockdown cells, which show resistance to induced mitochondrial fragmentation.

What are the optimal fixation and permeabilization conditions for CCDC51 antibody immunostaining?

For optimal CCDC51 antibody immunostaining, standard paraformaldehyde fixation (4% PFA for 15-20 minutes at room temperature) followed by permeabilization with a mild detergent such as 0.1-0.2% Triton X-100 has proven effective in multiple cell types including fibroblasts, HeLa, and U2OS cells . When working with retinal tissues, similar fixation protocols work well, though tissue-specific optimizations may be necessary. For co-staining experiments, ensure compatibility between fixation/permeabilization conditions for all target proteins. When combining CCDC51 antibody staining with mitochondrial dyes like MitoTracker, remember that some dyes require live-cell incubation prior to fixation. Following permeabilization, adequate blocking (typically with 5% normal serum) is essential to minimize non-specific binding of the CCDC51 antibody.

How can I differentiate between the discrete localization patterns of CCDC51 within mitochondria?

Differentiating between CCDC51's various localization patterns within mitochondria requires high-resolution imaging techniques and careful quantitative analysis. Confocal microscopy with z-stack acquisition is essential for capturing CCDC51's non-uniform distribution throughout individual mitochondria, including its occasional concentration in discrete focal structures . For optimal visualization, combine CCDC51 antibody staining with markers for different mitochondrial compartments (outer membrane: TOMM20; matrix: HSP60) to precisely map its relative position .

Super-resolution microscopy techniques such as STED or STORM can provide enhanced resolution of these distribution patterns. Quantitative image analysis should include measurements of signal intensity distribution, colocalization coefficients with various mitochondrial markers, and identification of enriched foci. In complementary experiments, GFP-tagged CCDC51 shows a similar patchy distribution pattern with occasional enrichment in discrete focal structures compared to general mitochondrial staining, validating antibody-based observations of the endogenous protein .

How does CCDC51 protein depletion affect mitochondrial morphology over time?

CCDC51 depletion causes a progressive and dynamic change in mitochondrial morphology that evolves over time. During acute depletion (approximately 72 hours post-siRNA treatment), mitochondria predominantly exhibit hyperfusion, resembling phenotypes observed in cells depleted of fission machinery like Drp1 . This initial phase occasionally includes the formation of small mitochondrial "nets" similar to those observed in yeast cells deficient for Dnm1 (the Drp1 homolog) .

With prolonged CCDC51 depletion (96-120 hours), there is a progressive shift toward more severe morphological abnormalities, with increasing prevalence of both net-like structures and distinctive lamellar mitochondria . By 120 hours post-knockdown, lamellar mitochondrial structures become the predominant phenotype. This temporal progression suggests CCDC51 plays a crucial role in maintaining normal mitochondrial fission dynamics, with its absence first causing reduced fission (leading to hyperfusion) and eventually triggering more dramatic structural rearrangements as cells attempt to compensate for prolonged protein loss .

What controls should be included when studying CCDC51's impact on mitochondrial fission?

When investigating CCDC51's role in mitochondrial fission, several critical controls should be included to ensure experimental validity and interpretability:

• Drp1 knockdown control: Depletion of Drp1 (using validated siRNA sequences like 5'-GACTTGTCTTCTTCGTAAATT-3') serves as a positive control for impaired fission, causing extensive mitochondrial hyperfusion and resistance to fission-inducing treatments .

• Time-course analysis: Include multiple time points (e.g., 48h, 72h, 96h, and 120h post-knockdown) to capture the progressive nature of mitochondrial morphology changes following CCDC51 depletion .

• Multiple cell types: Confirm phenotypes in different cell lines (e.g., U2OS and HeLa) to establish the generality of CCDC51's function .

• Fission induction controls: When testing fission dynamics, include both untreated and BAPTA-AM-treated conditions at multiple time points (e.g., 10 and 30 minutes) to assess the kinetics of fission impairment .

• Complementation control: Express siRNA-resistant CCDC51 (or GFP-CCDC51) in knockdown cells to confirm phenotype rescue, verifying specificity of the observed effects .

• Multiple knockdown approaches: Compare phenotypes between transient (siRNA) and stable (CRISPRi) CCDC51 depletion methods to distinguish acute from chronic effects .

What are the most informative mitochondrial markers to use alongside CCDC51 antibodies?

For comprehensive analysis of CCDC51 localization and function, combining multiple mitochondrial markers targeting different submitochondrial compartments provides the most informative results:

When performing co-immunostaining experiments, ensure antibodies are raised in different species to allow simultaneous detection with species-specific secondary antibodies. For optimal results in high-resolution imaging, sequential staining protocols may be necessary to minimize cross-reactivity .

How is CCDC51 expression pattern altered in retinal disorders?

In the context of inherited retinal disorders (IRDs), particularly rod-cone dystrophy (RCD), CCDC51 expression and function appear critically important for photoreceptor health. Under normal conditions, CCDC51/MITOK shows strong immunolocalization in the inner segments of both rod and cone photoreceptors, corresponding to areas with high mitochondrial concentration . In the reported case of RCD associated with a homozygous frameshift variant in CCDC51 (c.244_246delins17 p.(Trp82Valfs*4)), the mutation is predicted to produce a truncated, non-functional CCDC51 protein .

This suggests that complete loss of functional CCDC51 in photoreceptors may disrupt normal mitochondrial dynamics and function, ultimately contributing to photoreceptor degeneration. While comprehensive expression pattern analyses in multiple retinal disorder models are still needed, this initial evidence points to CCDC51 as a critical mitochondrial protein whose absence specifically affects photoreceptor cells despite its ubiquitous expression . Future immunohistochemical studies using CCDC51 antibodies in various retinal disease models would help elucidate whether expression patterns change prior to or during degeneration processes.

What methodological approaches are most effective for studying CCDC51 in retinal tissues?

For comprehensive analysis of CCDC51 in retinal tissues, multiple complementary methodological approaches are recommended:

• Transcriptomic analysis: Utilize RNA sequencing or qPCR to quantify CCDC51 transcript levels across different retinal cell types and layers. This approach has confirmed CCDC51 expression in human retina .

• Immunohistochemistry: Both fluorescent antibody staining and horseradish peroxidase-based methods have successfully demonstrated CCDC51 localization in retinal sections from multiple species including human, non-human primate, and mouse retinas .

• Co-localization studies: Combine CCDC51 antibodies with markers for specific retinal cell types (e.g., rhodopsin for rods, cone opsins for cones) and mitochondrial markers (e.g., ATP synthase) to precisely map expression patterns .

• Electron microscopy: For ultrastructural analysis of CCDC51's association with mitochondria in photoreceptor inner segments, immunogold labeling with CCDC51 antibodies can provide nanometer-scale resolution.

• Ex vivo retinal explants: For functional studies, retinal explant cultures combined with viral-mediated CCDC51 knockdown or overexpression can help assess acute effects on photoreceptor survival and mitochondrial morphology.

• Animal models: Development of CCDC51 knockout or knockin mouse models would facilitate in vivo studies of retinal phenotypes associated with CCDC51 dysfunction.

How can I effectively analyze CCDC51's role in mitochondrial pore formation?

Investigating CCDC51's role as a pore-forming subunit of mitochondrial K(ATP) channels requires specialized electrophysiological and biochemical approaches:

• Patch-clamp electrophysiology: Using isolated mitochondria or mitoplasts (mitochondria with the outer membrane removed), measure K+ currents in the presence and absence of CCDC51 (via knockout, knockdown, or antibody-mediated inhibition).

• Reconstitution assays: Purify CCDC51 protein and reconstitute it into lipid bilayers to directly measure its channel-forming capacity and characterize its electrophysiological properties.

• Proximity labeling: Employ BioID or APEX2 proximity labeling fused to CCDC51 to identify interacting partners involved in pore complex formation.

• Structure-function analysis: Generate and test CCDC51 mutants with alterations in potential pore-forming regions (particularly in the transmembrane domains) to identify critical residues for channel function.

• Mitochondrial membrane potential assays: Use potentiometric dyes (e.g., TMRM, JC-1) to assess how manipulation of CCDC51 levels affects mitochondrial membrane potential in response to K+ flux modulators.

• Pharmacological approaches: Apply known K(ATP) channel modulators (openers and blockers) and assess their effects on mitochondrial function in the presence and absence of CCDC51 to confirm its role in channel activity.

How might the evolutionary conservation between human CCDC51 and yeast Mdm33 inform experimental design?

The functional conservation between human CCDC51 and yeast Mdm33 provides a valuable evolutionary framework for designing experiments. Both proteins appear to be conserved mediators of mitochondrial morphology, with similar roles in promoting efficient mitochondrial fission . This conservation can inform experimental design in several ways:

• Complementation studies: Testing whether human CCDC51 can rescue phenotypes in Δmdm33 yeast cells would confirm functional conservation and identify domains critical for this shared function.

• Structure-function analysis: Comparing protein domains and motifs between CCDC51 and Mdm33 can highlight evolutionarily conserved regions likely to be functionally significant, guiding the design of targeted mutations.

• Interaction partners: Identifying Mdm33-interacting proteins in yeast can suggest potential CCDC51 interaction partners in human cells that may have been conserved throughout evolution.

• Stress response studies: Since both proteins influence mitochondrial dynamics during stress conditions (e.g., Mdm33's role in sodium azide-induced fission), parallel experiments examining CCDC51's function during cellular stress in human cells can reveal conserved regulatory mechanisms .

• Combined model approaches: Using both yeast and human cell models in parallel allows researchers to leverage the experimental advantages of each system—yeast for genetic manipulations and high-throughput screens, human cells for direct disease relevance.

What methodological approaches can resolve the temporal dynamics of CCDC51's impact on mitochondrial morphology?

To resolve the temporal dynamics of CCDC51's effects on mitochondrial morphology, several advanced methodological approaches are recommended:

• Inducible knockdown/knockout systems: Use doxycycline-inducible shRNA or CRISPRi systems to achieve temporal control over CCDC51 depletion, enabling precise monitoring of morphological changes from the earliest stages .

• Live-cell imaging: Implement time-lapse confocal or spinning disk microscopy of cells expressing mitochondrial markers (e.g., mito-GFP or mito-DsRed) during controlled CCDC51 depletion to capture morphological transitions in real time.

• Conditional protein degradation: Apply auxin-inducible or PROTAC-based rapid protein degradation systems targeting CCDC51 to achieve acute protein loss without the lag associated with transcriptional/translational inhibition.

• Photoactivatable CCDC51 inhibitors: Develop optogenetic tools to acutely inhibit CCDC51 function in specific mitochondrial subpopulations, allowing spatial and temporal control.

• Correlative light-electron microscopy: Combine live-cell imaging with subsequent electron microscopy of the same cells to connect dynamic events observed in real-time with ultrastructural changes.

• Quantitative morphometric analysis: Apply machine learning-based image analysis to quantify multiple parameters of mitochondrial morphology (length, branching, area, perimeter-to-area ratio) at defined time intervals following CCDC51 manipulation .

How can CCDC51 antibodies be utilized to investigate potential therapeutic approaches for mitochondrial disorders?

CCDC51 antibodies can serve as valuable tools in developing and evaluating therapeutic approaches for mitochondrial disorders, particularly those affecting mitochondrial dynamics and retinal health:

• Biomarker development: CCDC51 antibodies can help establish whether altered CCDC51 levels, localization, or post-translational modifications correlate with disease progression in patient samples, potentially serving as diagnostic or prognostic biomarkers.

• Therapeutic screening platforms: High-content imaging assays using CCDC51 antibodies can identify compounds that normalize mitochondrial morphology in cellular models of CCDC51 dysfunction or related mitochondrial fission defects.

• Gene therapy validation: For retinal disorders linked to CCDC51 mutations, antibodies can verify successful protein expression following gene therapy approaches in preclinical models.

• Protein-protein interaction targeting: CCDC51 antibodies can help characterize interaction interfaces between CCDC51 and other mitochondrial dynamics proteins, identifying potential sites for therapeutic intervention with small molecules or peptides.

• Cell-based assays: Using CCDC51 antibodies to monitor protein levels and localization can help assess the efficacy of antisense oligonucleotides or splice-modulating therapies designed to correct specific CCDC51 mutations.

• Mitochondrial delivery systems: For approaches involving delivery of therapeutic agents to mitochondria, CCDC51 antibodies can confirm whether treatments successfully reach their intended submitochondrial compartment.