MYO18A Antibody

What is MYO18A and what is its biological significance?

MYO18A encodes the protein 'myosin XVIIIA' in humans, a member of the unconventional myosin family. It may also be known by alternative names including MAJN, MYSPDZ, SP-R210, SPR210, and SP-A receptor subunit SP-R210 alphaS. Structurally, the protein is approximately 233.1 kilodaltons in mass . Recent research has identified MYO18A as a novel regulator in B cell differentiation and humoral immunity, where it functions as a checkpoint regulator that limits naïve B cell and immunoglobulin levels while also restricting antigen-induced humoral immunity . Beyond this immunological role, MYO18A is involved in stabilization and organization of the actin cytoskeleton, suggesting it has multifunctional cellular roles that continue to be elucidated through ongoing research .

Which species are typically studied with MYO18A antibodies and why?

MYO18A antibodies typically show reactivity with human, mouse, and rat samples, as confirmed in multiple validation studies . The conservation of MYO18A across these species makes them valuable models for comparative functional studies. Based on gene homology, canine and monkey orthologues may also be studied . Mouse models are particularly important in MYO18A research as demonstrated by B cell-conditional Myo18A-deficient mice studies that have revealed crucial insights into its regulatory functions in the immune system . Cross-species analysis is essential for understanding the evolutionary conservation of MYO18A's functions and validating research findings across different model systems.

What are the typical molecular weights observed for MYO18A in experimental settings?

While the calculated molecular weight of MYO18A is 233 kDa based on protein sequence analysis, researchers commonly observe bands at approximately 230 kDa and 190 kDa in Western blot applications . This discrepancy between calculated and observed molecular weights may result from post-translational modifications, alternative splicing, or proteolytic processing. When troubleshooting experimental results, researchers should anticipate potential variation in band appearance depending on cell or tissue type, sample preparation methods, and electrophoresis conditions. Proper positive controls using validated cell lines such as Jurkat, HeLa, K-562, Neuro-2a, or NIH/3T3 cells are essential for accurate band identification and interpretation .

Which experimental applications are validated for MYO18A antibodies?

MYO18A antibodies have been validated for multiple experimental applications, including Western Blot (WB), Immunoprecipitation (IP), Immunofluorescence (IF), Flow Cytometry (FC), Immunohistochemistry (IHC), and ELISA . The versatility of these antibodies enables comprehensive investigation of MYO18A's expression, localization, and function. For Western blot applications, researchers have successfully detected MYO18A in various cell lines including Jurkat, HeLa, K-562, Neuro-2a, and NIH/3T3 cells . For immunohistochemical applications, mouse heart tissue and skeletal muscle tissue have shown positive reactivity. Each application requires specific optimization of antibody dilution and experimental conditions to achieve optimal signal-to-noise ratios and reliable results.

What are the recommended dilutions and optimization strategies for MYO18A antibodies in different applications?

Optimal dilutions vary by application and should be empirically determined for each experimental system. For Western blot applications, a dilution range of 1:1000-1:6000 is typically recommended . For immunoprecipitation, 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate is suggested . Immunohistochemistry applications generally require dilutions between 1:500-1:2000 . For all applications, researchers should perform titration experiments to determine the optimal antibody concentration that maximizes specific signal while minimizing background. Sample-dependent variables such as target protein abundance, tissue fixation method, and detection system sensitivity will influence the optimal dilution. For IHC applications specifically, antigen retrieval methods significantly impact results, with TE buffer pH 9.0 often recommended, though citrate buffer pH 6.0 may serve as an alternative .

How should researchers validate the specificity of MYO18A antibodies in their experimental systems?

Rigorous validation of antibody specificity is essential for reliable research outcomes. Multiple approaches should be implemented:

• Positive and negative controls: Include cell lines known to express MYO18A (such as Jurkat, HeLa, K-562) as positive controls, and compare with tissues or cells where expression is absent or reduced .

• Genetic validation: Utilize knockout or knockdown models where MYO18A expression is genetically ablated or reduced. The antibody should show corresponding reduction or absence of signal in these samples .

• Peptide competition assays: Pre-incubation of the antibody with its specific immunizing peptide should eliminate specific binding.

• Multiple antibody validation: Confirm results using antibodies from different suppliers or those targeting different epitopes of MYO18A.

• Molecular weight verification: Ensure detected bands correspond to the expected molecular weights of 230 kDa and 190 kDa .

Published literature reports using MYO18A antibodies in knockout/knockdown studies provide additional validation references and should be consulted when interpreting experimental results .

How does MYO18A regulate B cell differentiation and what experimental approaches demonstrate this function?

MYO18A functions as a negative regulator of B cell differentiation through multiple mechanisms. B cell-conditional Myo18A-deficient mice exhibit expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs, indicating that MYO18A normally constrains B cell development and homeostasis . Experimentally, this has been demonstrated through flow cytometric analysis of B cell populations in Myo18A knockout versus control mice, revealing altered proportions of B cell subsets.

Further evidence comes from in vitro stimulation assays with TLR7 and BCR ligands, which demonstrated enhanced differentiation capacity of Myo18A-deficient B cells . Analysis of antibody-secreting cells (ASCs) in spleen and bone marrow compartments revealed increased splenic ASCs in Myo18A-deficient mice compared to controls, while bone marrow plasma cells remained similar . These findings collectively support experimental approaches combining genetic models (conditional knockout mice), flow cytometry for cellular phenotyping, and functional assays measuring B cell differentiation and antibody secretion.

What impact does MYO18A deficiency have on antibody production and autoimmunity?

MYO18A deficiency leads to a progressive dysregulation of antibody production characterized by distinct temporal phases. Initially, MYO18A-deficient mice display serum IgM hyperglobulinemia with increased numbers of splenic IgM-secreting cells . As these mice age, they transition to IgG1 hyperglobulinemia and develop autoantibodies against self-antigens, particularly showing increased antibodies to the lupus-associated Sm autoantigen .

This progression suggests MYO18A plays an important role in preventing inappropriate antibody responses. Mechanistically, this may be linked to the expansion of CD5+ B1a B cells observed in MYO18A-deficient mice, as these cells are known to produce natural IgM and can express autoreactive B cell receptors (BCRs) . Despite developing autoantibodies, these mice do not exhibit overt clinical manifestations of autoimmune disease such as weight loss, dermatitis, or joint inflammation . These findings highlight the complex relationship between antibody dysregulation and clinical autoimmunity, suggesting additional checkpoints may prevent progression to symptomatic disease.

How does MYO18A deficiency affect antigen-specific immune responses to viral antigens?

MYO18A deficiency enhances antigen-specific immune responses to viral antigens, as demonstrated in studies using inactivated influenza virus. When immunized with UV-inactivated PR8 influenza virus, MYO18A-deficient mice developed significantly higher levels of both anti-hemagglutinin (HA) IgM and anti-HA IgG antibodies compared to control mice . More importantly, these antibodies demonstrated superior functional capacity, with sera from MYO18A-deficient mice showing higher neutralization potency against live PR8 virus in MDCK cell infection assays .

The enhanced antibody response was associated with persistent accumulation of antigen-specific germinal center B cells and increased antigen-specific bone marrow plasma cells . These findings highlight MYO18A's role as a negative regulator of antigen-specific humoral immunity. The experimental approach combining immunization protocols, measurement of antigen-specific antibody titers by ELISA, functional neutralization assays, and analysis of germinal center dynamics provides a comprehensive methodology for investigating regulatory factors in vaccine responses.

What are the molecular mechanisms through which MYO18A regulates B cell function?

The molecular mechanisms underlying MYO18A's regulatory functions in B cells remain incompletely understood and represent an active area of investigation. As an unconventional myosin family member, MYO18A likely influences cytoskeletal organization and may regulate critical processes such as B cell receptor signaling, antigen internalization, or cellular migration . Given that B cell receptor signaling and trafficking are intimately connected with the actin cytoskeleton, MYO18A may serve as a link between cytoskeletal dynamics and immunological signaling pathways.

Experimental approaches to elucidate these mechanisms should include:

• Immunoprecipitation studies to identify MYO18A binding partners in B cells

• Phosphoproteomic analysis comparing signaling pathway activation in wild-type versus MYO18A-deficient B cells

• Live cell imaging to visualize cytoskeletal dynamics during B cell activation

• Analysis of antigen internalization and processing efficiency

• Investigation of transcriptional changes induced by MYO18A deficiency

These approaches would help delineate whether MYO18A's regulatory effects are mediated through direct influence on signaling pathways or indirectly through cytoskeletal reorganization affecting cellular processes.

How can MYO18A-related findings inform therapeutic approaches for autoimmune disorders?

The identification of MYO18A as a negative regulator of B cell differentiation and antibody production suggests potential therapeutic applications in conditions characterized by aberrant B cell activation and autoantibody production. The observed development of autoantibodies in MYO18A-deficient mice, particularly against lupus-associated antigens, provides a mechanistic link to human autoimmune disorders .

Therapeutic strategies might include:

• Enhancing MYO18A function: Developing compounds that enhance MYO18A activity or expression could potentially dampen excessive antibody responses in autoimmune conditions.

• Targeting downstream pathways: Identifying and modulating the signaling pathways regulated by MYO18A might provide more specific therapeutic targets.

• B cell subset modulation: Since MYO18A deficiency leads to expansion of specific B cell subsets, particularly CD5+ B1a B cells , targeting these populations might help control autoimmunity.

• Vaccine adjuvant development: Conversely, temporary inhibition of MYO18A function might enhance vaccine responses, as MYO18A-deficient mice develop more potent neutralizing antibodies against viral antigens .

Research must balance potential benefits against risks, as complete inhibition of MYO18A might promote autoimmunity even while enhancing desired immune responses.

How might post-translational modifications affect MYO18A function in different cellular contexts?

Post-translational modifications (PTMs) likely play critical roles in regulating MYO18A function across different cellular contexts. The discrepancy between calculated (233 kDa) and observed (230 kDa and 190 kDa) molecular weights suggests MYO18A undergoes significant processing or modification . Several research approaches can address the impact of PTMs on MYO18A function:

• Mass spectrometry analysis: To identify specific modifications including phosphorylation, ubiquitination, SUMOylation, or proteolytic processing.

• Site-directed mutagenesis: Generating mutants at potential modification sites to assess functional consequences.

• Stimulus-dependent modification: Analyzing how B cell activation signals affect MYO18A modification patterns.

• Tissue-specific modification patterns: Comparing PTM profiles across different tissues and cell types where MYO18A is expressed.

• Interaction proteomics: Determining how PTMs affect MYO18A's interaction with binding partners.

Understanding these modifications could reveal regulatory mechanisms controlling MYO18A's function in different cellular contexts and potentially explain tissue-specific phenotypes observed in MYO18A-deficient models.

What are common challenges in MYO18A detection and how can they be addressed?

Detection of MYO18A presents several technical challenges that researchers should anticipate and address:

• High molecular weight detection: At 230-233 kDa, MYO18A requires extended gel running times and optimized transfer conditions for Western blot applications. Use low percentage gels (6-8%), extend transfer times, and consider specialized transfer buffers for high molecular weight proteins.

• Multiple isoforms: The observation of both 230 kDa and 190 kDa bands indicates potential isoforms or processed variants . Verify which form predominates in your experimental system and confirm specificity through knockout controls.

• Low abundance in certain tissues: Expression levels vary significantly across tissues. Enhance detection by increasing protein loading, using more sensitive detection substrates, or employing signal amplification techniques.

• Antibody cross-reactivity: Validate antibody specificity through knockout/knockdown controls or peptide competition assays.

• Fixation-sensitive epitopes in IHC: Test multiple fixation methods and antigen retrieval protocols. Both TE buffer pH 9.0 and citrate buffer pH 6.0 have been successful for MYO18A IHC applications .

Maintaining detailed records of optimization experiments will facilitate reproducible protocols tailored to specific experimental systems.

What controls are essential for interpreting MYO18A antibody experimental results?

Rigorous experimental controls are critical for reliable interpretation of MYO18A antibody results:

• Positive tissue/cell controls: Include samples known to express MYO18A, such as Jurkat, HeLa, K-562, Neuro-2a, or NIH/3T3 cells for Western blot applications, or mouse heart tissue and skeletal muscle tissue for IHC .

• Genetic controls: When available, include MYO18A knockout or knockdown samples. Literature reports document the use of MYO18A antibodies in at least 4 knockout/knockdown studies .

• Loading controls: For Western blot applications, include appropriate loading controls matched to the cellular compartment where MYO18A is expected (typically cytoskeletal or membrane fractions).

• Secondary antibody controls: Include samples processed with secondary antibody only to identify non-specific binding.

• Dilution series: When quantifying MYO18A levels, include a dilution series of positive control samples to establish the linear detection range.

• Isotype controls: For flow cytometry or immunofluorescence applications, include appropriate isotype controls matched to the MYO18A antibody.

These controls allow for confident interpretation of experimental results and troubleshooting of unexpected outcomes.

How can researchers optimize experimental design for studying MYO18A in B cell function?

Designing experiments to study MYO18A's role in B cell function requires careful consideration of several factors:

• B cell isolation techniques: Use gentle isolation methods that preserve native cytoskeletal architecture, as harsh isolation procedures may disrupt MYO18A's interactions with the actin cytoskeleton.

• B cell subset consideration: Given the differential expansion of B cell subsets in MYO18A-deficient mice , analyze distinct B cell populations separately (follicular B cells, marginal zone B cells, B1 cells) rather than total B cells.

• Temporal dynamics: MYO18A-deficient mice show age-dependent progression from IgM to IgG1 hyperglobulinemia , necessitating time-course studies rather than single timepoint analyses.

• Activation context specificity: Compare multiple B cell activation pathways (BCR stimulation, TLR activation, CD40 signaling) as MYO18A may differentially regulate distinct activation pathways.

• In vivo versus in vitro approaches: Complement in vitro B cell stimulation assays with in vivo immunization models to capture the complete physiological context.

• Functional readouts: Include multiple readouts beyond antibody production, such as proliferation, apoptosis, differentiation markers, and signaling pathway activation.

• Cell migration and adhesion: Given MYO18A's role in cytoskeletal organization, assess B cell migration, adhesion, and interaction with other immune cells.

This comprehensive experimental approach will provide deeper insights into MYO18A's multifaceted roles in B cell biology and adaptive immunity.