Recombinant Human Coiled-coil domain-containing protein 51 (CCDC51)

What is the basic structure and localization of CCDC51?

CCDC51 is a 411-amino acid protein that contains an N-terminal mitochondrial targeting sequence (MTS) and two transmembrane (TM) domain segments interspersed with predicted coiled-coil domains . Structurally, it shares similarities with the yeast protein Mdm33 (455 amino acids), despite limited sequence homology . CCDC51 localizes to the inner mitochondrial membrane (IMM), with its coiled-coil domains facing both the mitochondrial matrix and the intermembrane space (IMS) . The protein contains specific structural motifs in its transmembrane domains, including glycine zipper motifs in TM1, which are critical for proper function and distribution within mitochondria .

What phenotypes are associated with CCDC51 depletion?

Depletion of CCDC51 through either stable CRISPRi knockdown or transient siRNA leads to distinct mitochondrial morphology defects. In stable knockdown cells, mitochondria form lamellar, sheet-like structures with non-uniform distribution of mitochondrial compartment markers . The matrix forms lariat ring structures similar to those observed in Δmdm33 yeast cells, while IMM and OMM markers appear as lamellae encapsulating the matrix marker . With acute depletion via siRNA, mitochondria primarily appear elongated and hyperfused, with occasional fenestrated "nets" of mitochondria resembling those found in cells deficient for Drp1, the key mitochondrial fission protein .

How does CCDC51 relate to mitochondrial dynamics?

CCDC51 plays a significant role in mitochondrial fission processes. Depletion of CCDC51 reduces the rate of mitochondrial fission events, though not eliminating them completely . Cells lacking CCDC51 show resistance to stress-induced mitochondrial fragmentation, similar to yeast Δmdm33 cells . Live-cell imaging reveals that CCDC51 localizes to discrete foci that mark approximately 36% of mitochondrial fission events, suggesting it functions as a positive effector for a subset of fission events . This spatial and temporal association with fission sites indicates CCDC51 works cooperatively with the canonical fission machinery including Drp1 .

What is the functional relationship between CCDC51 and the mitochondrial fission machinery?

CCDC51 appears to function as a positive effector of Drp1-mediated mitochondrial fission rather than an essential component of all fission events. Overexpression of CCDC51 promotes its accumulation at discrete sites and leads to Drp1-dependent fragmentation of the mitochondrial network . Remarkably, the majority of GFP-CCDC51 foci co-localize with Drp1 and MFF (a Drp1 receptor on the outer mitochondrial membrane), indicating that elevated CCDC51 levels promote the local recruitment of mitochondrial fission machinery . This relationship is specific, as CCDC51 foci show substantially less co-localization with mitochondrial DNA nucleoids than with Drp1 or MFF . The timing is also significant, as CCDC51 focal signals commonly disperse simultaneously with the completion of fission events (64% of events), suggesting a coordinated process .

How do the structural domains of CCDC51 contribute to its function?

Structure-function analysis reveals distinct roles for CCDC51's domains:

The IMS-localized coiled-coil is required for CCDC51's ability to rescue mitochondrial morphology defects, while the matrix-localized coiled-coil is dispensable . Mutations in the first TM domain, particularly in its glycine zipper motif, interfere with proper protein distribution and function . This suggests that IMS or TM domains may promote coupling of CCDC51 with mitochondrial fission machinery on the OMM, potentially through intermediary protein-protein interactions .

How do CCDC51-deficient cells respond to stress-induced mitochondrial fission?

CCDC51-depleted cells show altered responses to stress-induced mitochondrial fission. When treated with BAPTA-AM, a cell-permeable Ca²⁺ chelator known to induce Drp1-dependent mitochondrial fragmentation, control cells exhibit complete fragmentation of their mitochondrial network within 30 minutes . In contrast, cells depleted of CCDC51 show a delay in stress-induced mitochondrial fragmentation, though fission is not completely blocked as it is in Drp1-depleted cells . This resistance to stress-induced fission is consistent with observations in yeast Δmdm33 cells, which are resistant to sodium azide-induced Dnm1-dependent mitochondrial fragmentation . The data suggest CCDC51 facilitates efficient mitochondrial fission under stress conditions, possibly by enhancing the recruitment or activity of the canonical fission machinery.

What are the recommended approaches for studying CCDC51 localization and dynamics?

For studying CCDC51 localization and dynamics, a multi-faceted approach combining genetic manipulation with advanced imaging techniques is recommended:

• Genetic tools: Utilize CRISPRi for stable depletion or siRNA for acute knockdown of CCDC51 . For localization studies, express fluorescently tagged constructs (e.g., GFP-CCDC51) at low levels to avoid overexpression artifacts .

• Imaging approaches: Employ live-cell time-lapse microscopy to capture dynamic processes such as mitochondrial fission events . Use MitoTracker staining in conjunction with fluorescently tagged proteins to simultaneously visualize mitochondrial morphology and protein localization .

• Multi-color imaging: To examine the spatial relationship between CCDC51 and other mitochondrial components, use compartment-specific markers such as mCherry-OMP25 for OMM, TIMM50-GFP for IMM, and mito-HaloTag for the matrix . For analyzing co-localization with fission machinery, co-express GFP-CCDC51 with mCherry-Drp1 or immunolabel for endogenous Drp1/MFF .

• Quantification methods: Measure mitochondrial fission rates on an individual cell basis by counting fission events per minute during live-cell imaging sessions . Analyze the frequency of CCDC51-marked fission events compared to the total number of observed fission events .

How should one design domain deletion/mutation studies for CCDC51?

When designing domain deletion/mutation studies for CCDC51, consider the following methodological approach:

• Domain identification: First, use bioinformatic tools to accurately predict transmembrane domains, coiled-coil regions, and other structural features . For CCDC51, this includes identifying the N-terminal MTS, two TM domains, and coiled-coil domains in both the matrix and IMS .

• Mutation strategy: For TM domains, target conserved features such as glycine zipper motifs (G207A, G211A, G215A, and G218A in TM1) or polar residues (S204A, S208A, S219A, and T220A in TM1-NP) . For coiled-coil domains, consider complete deletion with replacement by flexible linkers, such as replacing amino acids 92-180 with an 8-amino acid Ser-Gly linker for the matrix coiled-coil, or amino acids 227-364 with a 12-amino acid Ser-Gly linker for the IMS coiled-coil .

• Rescue experiments: Express wild-type or mutant GFP-CCDC51 in CCDC51-depleted cells and assess rescue of mitochondrial morphology defects . Quantify the percentage of cells with tubular, intermediate, or lamellar mitochondrial morphology to determine rescue efficiency .

• Localization analysis: Examine whether mutations affect the subcellular distribution of CCDC51, particularly focusing on whether the protein forms distinct foci or distributes uniformly along mitochondria .

What techniques are effective for analyzing mitochondrial morphology in CCDC51 studies?

Effective techniques for analyzing mitochondrial morphology in CCDC51 studies include:

• Fluorescent staining approaches: Use MitoTracker dyes for live-cell imaging or immunolabeling for fixed-cell analysis . For comprehensive analysis, employ markers for different mitochondrial compartments (OMM, IMM, and matrix) to detect compartment-specific alterations .

• Morphology classification: Categorize mitochondrial morphology into distinct phenotypes (e.g., tubular, intermediate, lamellar/sheet-like) based on visual assessment, allowing for quantitative comparison between conditions .

• Stress induction assays: Apply agents like BAPTA-AM to induce mitochondrial fragmentation and assess the kinetics of morphological changes at different time points (e.g., 10 min, 30 min) . This approach reveals differences in the responsiveness of the mitochondrial network to fission stimuli .

• Dynamic analysis: Perform time-lapse imaging to measure mitochondrial fission rates, calculating events per minute per cell . This provides a quantitative measure of mitochondrial dynamics beyond static morphology .

• Co-visualization techniques: For investigating relationships between morphology and specific proteins or mtDNA, use dual-labeling approaches such as MitoTracker with PicoGreen (for mtDNA) or fluorescently tagged proteins .

How should researchers interpret changes in mitochondrial fission rates in CCDC51-modified systems?

When interpreting changes in mitochondrial fission rates in CCDC51-modified systems, researchers should consider multiple perspectives:

• Quantitative versus qualitative effects: CCDC51 depletion reduces but does not eliminate mitochondrial fission events, suggesting it facilitates a subset of fission events rather than being absolutely required for all fission . The ~32% reduction in fission frequency in CCDC51 CRISPRi cells correlates closely with the observation that ~36% of fission events are marked by GFP-CCDC51 foci in wild-type cells .

• Temporal considerations: Distinguish between acute and chronic effects of CCDC51 depletion. Acute depletion via siRNA primarily leads to mitochondrial hyperfusion, while long-term depletion results in the formation of lamellar structures . This suggests that hyperfusion may be the primary response, with lamellar structures developing secondarily over time .

• Comparison with canonical fission machinery: Compare CCDC51 depletion phenotypes with those of established fission factors like Drp1. While Drp1 depletion nearly eliminates all fission events, CCDC51 depletion has a partial effect, indicating distinct roles in the fission process .

• Stress-induced versus steady-state fission: Analyze both basal fission rates and responses to fission-inducing stimuli like BAPTA-AM . CCDC51-depleted cells show delayed response to stress-induced fragmentation, suggesting a role in efficient stress response .

What considerations are important when analyzing CCDC51 co-localization with mitochondrial fission machinery?

When analyzing CCDC51 co-localization with mitochondrial fission machinery, researchers should consider:

• Quantitative assessment: Determine the percentage of CCDC51 foci that co-localize with Drp1/MFF and compare with appropriate controls . In overexpression studies, the vast majority of GFP-CCDC51 foci co-localize with Drp1 and MFF foci, indicating a non-random association .

• Temporal dynamics: Examine the timing of CCDC51 focal accumulation relative to the recruitment of Drp1 and completion of fission . The observation that CCDC51 foci often disperse simultaneously with fission completion (64% of events) suggests coordinated timing .

• Specificity controls: Compare co-localization patterns with functionally unrelated structures like mtDNA nucleoids . CCDC51 foci show substantially less co-localization with mtDNA than with Drp1 or MFF, indicating specificity rather than random association .

• Expression level considerations: Distinguish between endogenous-like and overexpression conditions. While low levels of GFP-CCDC51 mark a subset of fission events, overexpression leads to increased focal accumulation and mitochondrial fragmentation .

• Mutant analysis: Use structure-function mutants to determine which domains are required for co-localization with fission machinery . This approach can reveal whether co-localization is mechanistically linked to CCDC51's function in fission .

What are the key unanswered questions about CCDC51 function in mitochondrial dynamics?

Several important questions remain regarding CCDC51's role in mitochondrial dynamics:

• Mechanistic coordination with OMM fission machinery: How does CCDC51, as an IMM protein, coordinate with Drp1 and adaptors on the OMM during fission events? The mechanisms by which these proteins communicate across two membrane bilayers remain to be elucidated .

• Interaction partners: What proteins interact with CCDC51, particularly in the IMS, to potentially bridge its function with the OMM fission machinery? The IMS protein Mdi1/Atg44 is required for Dnm1-mediated fission in yeast, raising questions about potential relationships between Mdi1/Atg44 and Mdm33, and whether similar interactions exist for CCDC51 in humans .

• Regulation of CCDC51 activity: What signals or modifications regulate CCDC51's association with fission sites? Understanding how CCDC51 is recruited to specific mitochondrial regions during fission events would provide insight into the coordination of fission across mitochondrial membranes .

• Functional heterogeneity: Why does CCDC51 mark only a subset of fission events? Determining whether CCDC51-associated fission events represent a functionally distinct subtype could reveal specialized roles in mitochondrial quality control or network remodeling .

What experimental approaches could address the coordination between CCDC51 (IMM) and Drp1 (OMM) during fission?

To investigate the coordination between CCDC51 in the IMM and Drp1 on the OMM during fission events, researchers might consider:

• Super-resolution microscopy: Employ techniques like STORM or PALM to visualize the nanoscale spatial relationship between CCDC51 and components of the OMM fission machinery during fission events .

• Proximity labeling approaches: Use split-BioID or APEX2-based proximity labeling to identify proteins that bridge the IMM and OMM at fission sites, potentially revealing molecular links between CCDC51 and Drp1 .

• Real-time correlation analysis: Perform dual-color live-cell imaging with tagged CCDC51 and Drp1 to analyze their temporal relationship during fission, determining whether one protein recruits the other or if they arrive independently .

• Systematic IMS protein analysis: Screen IMS-localized proteins for their impact on CCDC51-Drp1 coordination, potentially identifying intermediaries that facilitate communication across mitochondrial membranes .

• In vitro reconstitution: Develop membrane systems that recapitulate aspects of mitochondrial fission to test direct or indirect interactions between CCDC51 and components of the OMM fission machinery .

• Domain-specific perturbation: Use the IMS coiled-coil deletion mutant of CCDC51, which fails to rescue morphology defects, to identify potential interaction partners that may be lost when this domain is removed .