ATAD3A Antibody, FITC conjugated

What is ATAD3A and why is it significant in cellular research?

ATAD3A is a mitochondrial inner membrane protein conserved in metazoans that has been associated with several critical mitochondrial functions, including nucleoid organization, cholesterol metabolism, and mitochondrial translation . Its significance stems from its scaffolding role in regulating mitochondrial inner membrane integrity and its interactions with multiple protein complexes fundamental for membrane organization . Deletion of ATAD3A causes embryonic lethality in various model organisms, highlighting its essential nature . Additionally, disease-causing mutations in ATAD3A have been identified, including duplications, deletions, and point mutations, making it relevant for both basic and translational research .

What is the specific subcellular localization of ATAD3A that researchers should consider when designing experiments?

ATAD3A displays a distinctive topology within mitochondria that's critical for experimental design. Immunological approaches have confirmed that the N-terminal region (amino acids 40-53) is accessible from the cytoplasm or intermembrane space, while the C-terminal region (amino acids 572-586) is located within the matrix . This unique orientation means ATAD3A spans both mitochondrial membranes, with the N-terminal domain outside the inner membrane and the C-terminal domain inside the matrix . Researchers should consider this dual localization when designing experiments, as it affects epitope accessibility in different experimental conditions.

What are the optimal fixation and permeabilization methods for ATAD3A immunofluorescence using FITC-conjugated antibodies?

For optimal ATAD3A immunofluorescence using FITC-conjugated antibodies, researchers should consider the unique topology of ATAD3A within mitochondrial membranes. Since the N-terminal region is accessible in the cytoplasm or intermembrane space while the C-terminal region is in the matrix , fixation and permeabilization protocols must be carefully optimized. For intact cell imaging, 4% paraformaldehyde fixation for 15-20 minutes at room temperature followed by permeabilization with 0.2-0.5% Triton X-100 generally provides good results. For isolated mitochondria preparations, milder permeabilization with 0.1% digitonin may better preserve the mitochondrial membrane structure while still allowing antibody access. When targeting the matrix-facing C-terminal epitopes, more robust permeabilization may be required. It's advisable to compare multiple fixation protocols (e.g., paraformaldehyde vs. methanol) in parallel to determine optimal conditions for specific experimental setups.

How should researchers optimize immunofluorescence protocols to detect ATAD3A oligomerization in neurodegenerative disease models?

To optimize detection of ATAD3A oligomerization in neurodegenerative disease models, researchers should implement several specialized approaches. First, sample preparation is crucial—non-reducing conditions (absence of β-mercaptoethanol) should be maintained to preserve oligomeric structures that increase in Alzheimer's disease models . When examining tissue samples, use a combination of immunohistochemistry with ATAD3A antibodies and co-localization with neuronal markers like NeuN to specifically assess neuronal ATAD3A levels, as demonstrated in AD patient and mouse model studies . For higher resolution analysis, proximity ligation assays (PLA) can be employed to detect ATAD3A interactions with MAM markers like FACL4, which showed approximately twofold increase in AD models . Additionally, when using cell models, researchers can incorporate chemical cross-linkers (such as bismaleimidohexane, BMH) to stabilize and enhance detection of transient oligomeric interactions . Finally, time-course experiments are recommended as ATAD3A oligomerization increases in a time- and dose-dependent manner upon exposure to factors like oligomeric Aβ peptides .

What controls are essential when using FITC-conjugated ATAD3A antibodies for co-localization studies with mitochondrial markers?

When conducting co-localization studies with FITC-conjugated ATAD3A antibodies and mitochondrial markers, several controls are essential for data integrity. First, include an isotype control antibody with FITC conjugation to assess non-specific binding. Second, implement single-color controls to establish proper compensation parameters and detect any bleed-through between channels. Third, use mitochondrial subcompartment markers as biological controls—Tom20 for outer membrane, Tim23 for inner membrane, and matrix proteins like HSP60—to verify ATAD3A's reported dual localization pattern . Include functional controls using ATAD3A knockdown or knockout samples where available, particularly neuron-specific conditional knockout models as referenced in studies . For advanced applications, consider preparing cells with mutant ATAD3A constructs (such as N-terminal deletion mutants like Atad3a dN50) which fail to interact with outer membrane components , providing important negative controls for localization specificity.

How can researchers utilize FITC-conjugated ATAD3A antibodies to investigate its role in mitophagy regulation?

To investigate ATAD3A's role in mitophagy regulation using FITC-conjugated antibodies, researchers should implement a multi-faceted approach targeting the ATAD3A-PINK1 axis. First, establish co-immunofluorescence protocols combining FITC-ATAD3A antibodies with markers for PINK1 and mitophagy components to visualize their spatial relationships in real-time. Research has demonstrated that ATAD3A serves as a bridging factor facilitating PINK1 transport from the outer to inner mitochondrial membrane , making this interaction a key target for investigation.

For functional studies, researchers can employ time-lapse imaging in cells with manipulated ATAD3A levels (knockdown or overexpression) to track mitophagic flux using the FITC-ATAD3A antibody alongside lysosomal markers like LAMP2a. Previous studies showed increased co-localization of mitochondrial markers with LAMP2a upon ATAD3A knockdown .

Additionally, implement quantitative analysis of mitochondrial mass using mitochondrial DNA-to-nuclear DNA ratio measurements alongside antibody-based visualization to correlate ATAD3A localization patterns with mitophagy activity. Consider comparing wild-type ATAD3A with mutants lacking either ATPase activity (E412Q) or the N-terminal domain (dN50) to distinguish structure-function relationships, as research shows the N-terminus but not ATPase activity is required for PINK1 processing .

What are the key considerations when designing experiments to study ATAD3A's interactions with membrane protein complexes using FITC-conjugated antibodies?

When studying ATAD3A's interactions with membrane protein complexes using FITC-conjugated antibodies, researchers must consider several critical factors. First, ATAD3A's topology spanning both mitochondrial membranes requires careful experimental design—the N-terminal region is accessible from the cytoplasm/intermembrane space while the C-terminal region resides in the matrix . This means epitope accessibility varies depending on membrane integrity and experimental conditions.

Second, researchers should design co-immunoprecipitation experiments followed by fluorescence microscopy to validate interactions identified through mass spectrometry approaches. Previous studies identified 280 potential ATAD3A interactors, including prohibitin complex components (PHB, PHB2, DNAJC19, STOML2) and other membrane organization machinery .

Third, implement proximity-dependent biotinylation assays (BioID) as complementary approaches to map proximity interactors to different ATAD3A domains, particularly the C-terminal region, which has proven effective in previous research .

Fourth, consider membrane integrity—ATAD3A interacts with mitochondrial channel components Tom40 and Tim23 , requiring experimental conditions that preserve these delicate membrane structures. Compare detergent-based and detergent-free extraction methods to identify optimal conditions for maintaining complex integrity.

Finally, include controls with ATAD3A mutants lacking specific domains to map interaction regions, as studies have shown the N-terminal 50 amino acids are crucial for interactions with Tom40 and PINK1 .

How can researchers troubleshoot non-specific binding when using FITC-conjugated ATAD3A antibodies?

To troubleshoot non-specific binding with FITC-conjugated ATAD3A antibodies, researchers should implement a systematic approach addressing several potential issues. First, optimize antibody concentration through careful titration experiments—excessive antibody concentrations frequently contribute to background fluorescence. Second, implement rigorous blocking protocols using 5-10% normal serum from the same species as secondary antibodies or BSA supplemented with 0.1-0.3% Triton X-100 to reduce non-specific binding.

Third, validate antibody specificity using appropriate controls including ATAD3A knockout or knockdown samples if available. Studies have utilized neuronal-specific conditional knockouts of ATAD3A and shRNA knockdown models which would serve as excellent negative controls.

Fourth, implement cross-adsorption of antibodies against tissues from other species if cross-reactivity is observed. For multi-labeling experiments, perform single-label controls to identify any potential cross-reactivity between detection systems.

Fifth, consider autofluorescence, particularly in tissues with high mitochondrial content or in neurodegenerative disease models where lipofuscin accumulation can interfere with FITC signals. Treatment with Sudan Black B (0.1-0.3%) after immunolabeling can reduce autofluorescence.

Finally, adjust imaging parameters carefully—photobleaching FITC briefly before capturing images can sometimes reduce background, and implementing spectral unmixing during confocal microscopy can help distinguish specific signals from autofluorescence.

How should researchers quantify changes in ATAD3A expression and localization in neurodegenerative disease models?

For quantifying changes in ATAD3A expression and localization in neurodegenerative disease models, researchers should employ multi-dimensional analysis approaches. First, distinguish between oligomeric and monomeric forms of ATAD3A by comparing reducing and non-reducing conditions during sample preparation, as ATAD3A oligomerization increases in Alzheimer's disease models and patient tissues .

Second, implement region-specific analysis, as studies have shown that ATAD3A oligomers are elevated in specific brain regions (cortex, hippocampus, and thalamus) of 5XFAD AD mice but not in other regions . This regional specificity correlates with Aβ aggregation patterns.

Third, quantify both immunodensity and distribution patterns. Research has demonstrated increased ATAD3A immunodensity specifically in NeuN-positive neurons in cortical layer IV-V, subiculum, and hippocampus of AD mouse brains .

Fourth, apply co-localization analysis with appropriate cellular markers—enrichment of ATAD3A has been observed in APP-immunopositive cells of AD patient and mouse cortex . Additionally, use proximity ligation assays (PLA) to quantify interactions between ATAD3A and mitochondria-associated ER membrane (MAM) markers like FACL4, which increase approximately twofold in AD models .

Finally, correlate ATAD3A localization changes with functional outcomes by pairing immunofluorescence data with assessments of mitochondrial function, as ATAD3A oligomerization impacts critical mitochondrial processes.

What are the best approaches for analyzing ATAD3A's role at mitochondria-associated ER membranes (MAMs) using imaging techniques?

To analyze ATAD3A's role at mitochondria-associated ER membranes (MAMs) using imaging techniques, researchers should implement a comprehensive strategy that captures both structural and functional aspects of these specialized contact sites. First, employ proximity ligation assays (PLA) using antibody pairs targeting ATAD3A and established MAM markers like FACL4 . This approach has successfully demonstrated increased ATAD3A-FACL4 interaction in Alzheimer's disease models, with approximately twofold more PLA-positive puncta in affected tissues .

Second, quantify both the number and size of interaction puncta, as research shows that PLA-positive puncta between IP3R3 and VDAC (indicating MAM tethering) and between ATAD3A and FACL4 are larger in AD models, suggesting altered MAM architecture .

Third, implement high-resolution microscopy techniques (STED, STORM, or SIM) to assess the spatial relationship between ATAD3A and other MAM components. Previous research using high-resolution microscopy demonstrated increased co-localization between IP3R3 and VDAC in cells exposed to oligomeric Aβ peptides .

Fourth, combine imaging with functional assays measuring calcium transfer or lipid trafficking between ER and mitochondria, as ATAD3A has been implicated in cholesterol metabolism and MAM integrity .

Finally, implement live-cell imaging approaches when possible to capture the dynamic nature of these contacts, particularly in response to cellular stressors known to affect MAM integrity or ATAD3A oligomerization.

How can researchers distinguish between mitochondrial subcompartment localization of ATAD3A using FITC-conjugated antibodies?

To distinguish between mitochondrial subcompartment localization of ATAD3A using FITC-conjugated antibodies, researchers should implement a multi-faceted approach combining selective permeabilization, super-resolution imaging, and biochemical fractionation validation. First, employ differential permeabilization protocols—0.001-0.002% digitonin selectively permeabilizes the outer membrane while leaving the inner membrane intact, allowing discrimination between intermembrane space and matrix localization of ATAD3A epitopes. Previous research has established that ATAD3A's N-terminal region is accessible from the cytoplasm or intermembrane space, while its C-terminal region resides in the matrix .

Second, implement co-localization analysis with well-characterized subcompartment markers—Tom20 (outer membrane), Tim23 (inner membrane translocase), and matrix proteins like HSP60. ATAD3A has been shown to interact with Tom40 and Tim23, serving as a bridging factor between these components .

Third, apply super-resolution microscopy techniques (STED, STORM) to resolve the precise spatial relationship between ATAD3A and membrane structures with nanometer resolution, overcoming the diffraction limit of conventional fluorescence microscopy.

Fourth, validate imaging results with biochemical approaches like protease protection assays on isolated mitochondria with intact or selectively disrupted membranes. Previous studies used limited proteolysis to demonstrate the accessibility of ATAD3A domains .

Finally, compare results with electron microscopy immunogold labeling of ATAD3A to achieve the highest possible resolution of subcompartment localization, providing definitive spatial information that complements the fluorescence-based approaches.

How should researchers design experiments to investigate ATAD3A's role in cholesterol metabolism using FITC-conjugated antibodies?

To investigate ATAD3A's role in cholesterol metabolism using FITC-conjugated antibodies, researchers should design a comprehensive experimental approach integrating imaging, genetic manipulation, and functional assays. First, establish co-localization experiments combining FITC-ATAD3A immunofluorescence with fluorescent cholesterol probes (e.g., filipin) to visualize spatial relationships between ATAD3A and cholesterol pools within mitochondria and at MAMs.

Second, implement genetic manipulation studies comparing wild-type conditions with ATAD3A knockdown, knockout, or overexpression models, as research has shown that ATAD3A can influence cholesterol accumulation through CYP46A1, an enzyme governing brain cholesterol clearance . ATAD3A heterozygous knockout has been shown to restore neuronal CYP46A1 levels and normalize brain cholesterol turnover in AD models .

Third, design time-course experiments to capture dynamic changes in ATAD3A localization following manipulations of cellular cholesterol (using statins, cyclodextrin, or cholesterol oxidase) to establish causal relationships between ATAD3A distribution and cholesterol homeostasis.

Fourth, quantify changes in mRNA and protein levels of cholesterol metabolism enzymes (particularly CYP46A1) following ATAD3A manipulation, correlating these changes with immunofluorescence patterns of ATAD3A distribution.

Finally, design experiments to assess mitochondrial membrane fluidity and potential in relation to ATAD3A and cholesterol levels, as cholesterol content significantly impacts mitochondrial membrane properties and function.

What experimental approaches can best address the contradictions in literature regarding ATAD3A's interaction with mitochondrial DNA?

To address contradictions in literature regarding ATAD3A's interaction with mitochondrial DNA, researchers should implement a multi-modal experimental strategy that directly examines this controversial relationship. First, design chromatin immunoprecipitation (ChIP) experiments using FITC-conjugated ATAD3A antibodies followed by qPCR or sequencing of mitochondrial DNA regions to determine specific vs. non-specific interactions. This approach addresses reports that the N-terminal domain (amino acids 44-247) interacts with mtDNA while considering contradictory findings that ATAD3A could not be cross-linked to nucleoids despite being found in purified nucleoid complexes .

Second, implement in situ proximity ligation assays (PLA) between ATAD3A and mtDNA-binding proteins like TFAM to visualize potential interactions within intact cells, avoiding artifacts that might occur in cell fractionation studies.

Third, design experiments using ATAD3A mutants lacking specific domains, particularly the N-terminal region reported to interact with mtDNA , to determine which regions are essential for any observed interactions with nucleoids.

Fourth, apply super-resolution microscopy techniques to precisely map the spatial relationship between ATAD3A and mtDNA at nanometer resolution, providing direct visual evidence of their physical proximity or separation.

Finally, implement functional studies measuring mtDNA maintenance, replication, or transcription in models with manipulated ATAD3A levels (knockdown/overexpression), correlating any observed phenotypes with direct interaction data to distinguish between direct and indirect effects of ATAD3A on mitochondrial DNA.

How can researchers effectively study ATAD3A's relationship with PINK1-dependent mitophagy using FITC-conjugated antibodies?

To effectively study ATAD3A's relationship with PINK1-dependent mitophagy using FITC-conjugated antibodies, researchers should implement a comprehensive experimental framework addressing the mechanistic interaction between these proteins. First, design co-immunofluorescence experiments to visualize the spatial and temporal relationships between ATAD3A and PINK1 under basal conditions and following mitophagy induction. Research has established that ATAD3A serves as a bridging factor facilitating PINK1 transport and processing .

Second, implement live-cell imaging with FITC-ATAD3A antibody fragments or complementary fluorescent protein fusion constructs to track dynamic changes during mitophagy, correlating ATAD3A distribution with mitochondrial elimination events.

Third, design genetic interaction studies combining ATAD3A and PINK1 manipulations. Research has demonstrated that deletion of PINK1 in ATAD3A-deficient mice significantly rescued the mitophagy defect and restored progenitor and HSC pools , providing a foundation for exploring this genetic relationship in other cell types.

Fourth, quantify mitophagy flux using established markers (LC3-II in mitochondrial fractions, co-localization of Tom20 with Lamp2a) in cells with normal and manipulated ATAD3A levels. Studies show that knockdown of ATAD3A significantly increased co-localization of mitochondrial and lysosomal markers .

Fifth, implement structure-function analyses using ATAD3A mutants—particularly comparing wild-type ATAD3A with the E412Q (ATPase-dead) and dN50 (N-terminus deletion) variants—as research shows the N-terminus but not ATPase activity is required for PINK1 interaction and processing .

Finally, design rescuing experiments to test whether restoration of wild-type ATAD3A or specific mutants can normalize mitophagy rates in ATAD3A-deficient backgrounds, providing causal evidence for its role in mitophagy regulation.