ELMOD3 Antibody

What is ELMOD3 and what cellular functions does it perform?

ELMOD3 is a member of the ELMOD family proteins that function as GTPase-activating proteins (GAPs). It specifically exhibits GAP activity against Arf-family small GTP binding proteins, particularly ARL2, which is associated with the regulation of actin cytoskeleton dynamics via intracellular signaling pathways . ELMOD3 plays crucial roles in cilia formation and function, particularly in protein transport to cilia, and has significant functions at the Golgi apparatus . It strongly interacts with the actin cytoskeleton, which is particularly important in specialized cells such as cochlear hair cells. Mutations in ELMOD3 have been associated with hearing loss, highlighting its physiological importance in auditory function .

How is ELMOD3 protein structured and what domains does it contain?

ELMOD3 contains a conserved ELMO domain of approximately 160 residues, which is also present in human ELMO proteins . The human ELMOD3 isoform b (NM_001135021.1) encodes a protein of 381 residues . The ELMO domain represents the sole functional domain in ELMOD3 and is responsible for its GAP activity . Recent structural analysis using AlphaFold has provided detailed models of human ELMOD3 protein structure . The Gly214 residue, which has been implicated in hearing loss when mutated to serine, is located in a helix of the ELMO domain and is critical for protein stability . This residue forms important intermolecular interactions that maintain the protein's structural integrity and functional capacity.

Where is ELMOD3 typically expressed in cells and tissues?

Wild-type ELMOD3 is expressed predominantly in the cytoplasm and plasma membrane of cells . In hair cells of developing rat cochlea, ELMOD3 expression has been detected in stereocilia, kinocilia, and cuticular plate, indicating its importance in auditory sensory cells . ELMOD3 co-localizes with F-actin in normal conditions, which is consistent with its role in cytoskeletal regulation . It is also present at the Golgi apparatus and involved in trafficking to cilia . Notably, ELMOD3 is expressed at relatively low levels in many cell types, including mouse embryonic fibroblasts (MEFs), making detection challenging without specialized techniques .

What are the best methods to detect ELMOD3 in different cell types?

For detecting ELMOD3 in experimental settings, several approaches can be employed with varying success rates:

Western blotting with validated antibodies is effective for detecting overexpressed ELMOD3, as demonstrated in studies using HEI-OC1 cells transfected with ELMOD3-Myc-Flag constructs . Immunofluorescence microscopy can be used to detect ELMOD3 localization in cells, though it works more reliably with overexpressed protein . For endogenous ELMOD3 detection, specialized cell types with higher natural expression should be selected, such as cochlear-derived cell lines or retinal cells . RT-PCR can confirm ELMOD3 mRNA expression but should be complemented with protein detection methods.

How can I validate the specificity of an ELMOD3 antibody?

Validating antibody specificity is critical for reliable ELMOD3 research. The recommended validation approach includes:

• Generate ELMOD3 knockout cell lines using CRISPR/Cas9 targeting upstream of the ELMO domain .

• Compare antibody signal between wild-type and knockout cells by Western blot to confirm specificity .

• Perform siRNA knockdown as an alternative validation method for temporary reduction in expression.

• Use overexpression of tagged ELMOD3 (e.g., with Myc-Flag tags) as a positive control .

• Test the antibody in multiple applications (Western blot, immunofluorescence, immunoprecipitation) to assess cross-reactivity and specificity across different experimental conditions .

This validation pipeline is essential as currently there are no antibodies specific to ELMOD3 with the requisite sensitivity to detect endogenous proteins in certain cell types like MEFs .

What cell lines express the highest levels of ELMOD3 for experimental purposes?

Selecting appropriate cell lines is crucial for successful ELMOD3 studies:

Researchers should consult proteomics databases to identify cell lines with high ELMOD3 expression . Retinal photoreceptor cells have been used successfully for studying ELMOD localization . HEI-OC1 and MDCK cells have been employed effectively for ELMOD3 studies, particularly for overexpression experiments . MEFs express ELMOD3 at levels so low that they challenge detection by standard methods and mass spectrometry, with Ct values ≥35 in RNA-seq data .

How does ELMOD3 contribute to cilia formation and function?

ELMOD3 plays a critical role in ciliary biology through several mechanisms:

ELMOD3 regulates protein trafficking to cilia, and its absence is associated with improper transportation of proteins to these structures . Knockout studies have demonstrated that ELMOD3 deletion leads to decreased ability of cells to form primary cilia and results in the loss of specific proteins from cilia . ELMOD3 affects ciliation through its GAP activity on ARL family GTPases, particularly ARL2 . Interestingly, ELMOD3 functions can be partially rescued by activating mutant expression of either ARL3 or ARL16, linking these GTPases to ELMOD3's actions in cilia formation .

Additionally, ELMOD3 shares functional overlap with ELMOD1 in cilia formation and maintenance, as demonstrated by similar phenotypes in single and double knockout cell lines .

What is the role of ELMOD3 in hearing and how do mutations affect auditory function?

ELMOD3 plays a crucial role in hearing through its expression in cochlear hair cells:

ELMOD3 is expressed in stereocilia, kinocilia, and cuticular plate in cochlear hair cells, all critical structures for sound transduction . The p.Gly214Ser mutation in ELMOD3 has been associated with autosomal dominant hearing loss . Molecular modeling and structure analysis indicate that this mutation introduces spatial clashes with adjacent Ala160 and Cys162 residues, disrupting intermolecular interactions and compromising protein stability .

Functional assays have revealed that the mutant protein exhibits altered subcellular localization, accumulating in the nucleus rather than the cytoplasm and plasma membrane . The mutant also fails to localize with F-actin and demonstrates increased degradation rates in cycloheximide chase assays . These alterations likely disrupt stereocilia morphology in hair cells, as Elmod3 knockout mice show morphological dysgenesis of stereocilia within cochlear hair cells .

How do ELMOD3 and ELMOD1 functionally overlap in cellular processes?

ELMOD1 and ELMOD3 share significant functional overlap:

Both ELMOD1 and ELMOD3 function at the Golgi and cilia, with deletion of either gene resulting in decreased ability to form primary cilia . These proteins are involved in trafficking proteins from Golgi to cilia, and phenotypes resulting from their deletion show strong similarities . The functional redundancy between ELMOD1 and ELMOD3 is highlighted by the observation that deletion of both genes (double knockout) produces similar, not additive, phenotypes to single knockouts .

Both proteins interact with ARL3 and ARL16 in their cellular functions, as evidenced by rescue experiments in which activating mutations in these ARL proteins reversed phenotypes in ELMOD1/3 knockout cells .

Why might my ELMOD3 antibody not detect the protein in Western blots?

Several factors can contribute to challenges in detecting ELMOD3 by Western blot:

Low endogenous expression levels of ELMOD3 in many cell types is a primary challenge, as noted in MEFs where protein levels are below detection by mass spectrometry despite confirmed mRNA expression . Available antibodies may lack sufficient sensitivity for endogenous detection, as there are currently no antibodies specific to ELMOD3 with the requisite sensitivity to detect endogenous proteins in MEFs .

Non-specific or cross-reactive antibodies can yield misleading results, necessitating proper validation against knockout controls . Improper sample preparation may affect protein extraction, particularly if the protein is associated with insoluble cytoskeletal components. Additionally, ELMOD3 may be subject to rapid degradation during sample processing, as suggested by the increased degradation rate observed for mutant ELMOD3 .

How can I improve immunofluorescence results with ELMOD3 antibodies?

For optimal immunofluorescence detection of ELMOD3:

• Consider using epitope-tagged ELMOD3 constructs (e.g., Myc-Flag tags) as demonstrated in studies with MDCK cells .

• Optimize fixation methods to preserve epitope accessibility – paraformaldehyde fixation has been successfully used in published studies .

• Include co-staining for relevant cellular structures (e.g., F-actin, Golgi markers) to assess co-localization and provide context for ELMOD3 localization .

• Use confocal microscopy for better resolution of subcellular localization.

• Include appropriate controls, including ELMOD3 knockout cells and secondary antibody-only controls.

When analyzing subcellular localization, be aware that mutations can dramatically alter localization patterns, as seen with the p.Gly214Ser mutant that accumulates in the nucleus rather than the cytoplasm and plasma membrane .

How should I interpret altered ELMOD3 localization patterns in disease models?

Changes in ELMOD3 localization can provide important insights into disease mechanisms:

When analyzing altered localization, compare with known mutants like p.Gly214Ser that show nuclear accumulation instead of the normal cytoplasmic and membrane localization . Assess co-localization with relevant cellular structures, particularly F-actin, as the p.Gly214Ser mutant shows significantly decreased co-localization with F-actin compared to wild-type protein .

Consider effects on protein stability and turnover, as altered localization may be associated with changes in protein degradation rates, as demonstrated in cycloheximide chase assays with the p.Gly214Ser mutant . Evaluate functional consequences on cellular processes such as cilia formation, which can be disrupted by ELMOD3 mutations or deletions . Finally, determine if mislocalization affects GAP activity against ARL2 or other targets, which could disrupt downstream signaling pathways .

What experimental approaches can resolve contradictory results about ELMOD3 function?

When faced with conflicting data about ELMOD3 function, consider these approaches:

Generate multiple knockout cell lines using different guide RNAs targeting different exons of ELMOD3 to confirm phenotypes, as demonstrated in studies using CRISPR/Cas9 editing . Perform rescue experiments with wild-type and mutant ELMOD3 to confirm specificity of observed phenotypes, which can provide strong evidence for causality .

Use multiple cell types to account for context-specific effects, as ELMOD3 function may vary between different cellular environments . Combine in vitro biochemical assays (e.g., GAP activity assays) with cellular studies to connect molecular function to cellular phenotypes . Apply complementary techniques to study the same biological process, such as combining protein stability assays with localization studies .

How can I quantify changes in ELMOD3 expression or stability accurately?

For accurate quantification of ELMOD3:

Use quantitative Western blotting with appropriate loading controls (e.g., β-actin) as demonstrated in cycloheximide chase assays . Apply RT-qPCR for transcript level analysis, but recognize that mRNA levels may not directly correlate with protein levels, especially for low-abundance proteins like ELMOD3 .

For protein stability measurements, conduct cycloheximide chase assays with multiple timepoints (e.g., 0, 2, 4, 6, and 8 hours) to track degradation rates, as was done for comparing wild-type and mutant ELMOD3 . Always include appropriate statistical analysis for experimental replicates to determine significance, as demonstrated in the p.Gly214Ser mutation study where significant differences in degradation rates were observed at 2 and 8 hours (p = 0.028 and p = 0.0072, respectively) .