MYL12B Human

What is MYL12B and what is its function in human cells?

MYL12B functions as a regulatory light chain of nonmuscle myosin II. The activity of nonmuscle myosin II is regulated through phosphorylation of regulatory light chains such as MYL12B (also known as MRLC2). This phosphorylation results in higher MgATPase activity and facilitates the assembly of myosin II filaments . As a regulatory subunit, MYL12B plays an important role in controlling both smooth muscle and nonmuscle cell contractile activity through its phosphorylation state .

What are the primary identifiers and characteristics of human MYL12B?

Human MYL12B has several key identifiers and characteristics that researchers should be aware of:

The protein functions as a regulatory component of the myosin II complex, with its activity primarily controlled through phosphorylation .

How does MYL12B relate to its paralogous proteins?

MYL12B is highly homologous to two other regulatory light chains (RLCs): MYL12A and MYL9. These three paralogs share significant sequence similarity and overlapping functions in regulating myosin II activity . The murine orthologs (Myl12a, Myl12b, and Myl9) have been shown to be required for maintaining the stability of myosin II and cellular integrity . Functionally, these paralogs exhibit a complex relationship with partial redundancy, as demonstrated by the fact that single knockdowns of MYL12A or MYL12B have minimal effects on cell viability in many contexts, while double knockdown can cause major alterations in cell structure not observed in isoform-specific knockdowns .

What is known about MYL12B phosphorylation?

MYL12B regulation occurs primarily through phosphorylation. There are two groups of residues on MYL12B that are phosphorylated by distinct kinases, resulting in contrasting effects on myosin II biophysical properties . In particular, phosphorylation at Thr18/Ser19 is especially important, as it enhances MgATPase activity and promotes the assembly of myosin II filaments . This phosphorylation mechanism is central to MYL12B's role in controlling cell contractility, as it modulates the activity state of the associated myosin II heavy chains.

Which tissue types express MYL12B?

Based on antibody validation studies, MYL12B has been detected in multiple tissue types:

This wide distribution reflects MYL12B's fundamental role in cellular processes across different tissue types .

What experimental approaches are most effective for studying MYL12B?

Multiple validated experimental approaches exist for studying MYL12B, each with specific recommendations:

When selecting antibodies, researchers should consider the specific epitopes recognized and whether they can distinguish between MYL12B and its highly homologous paralogs. The antibody product referenced in the search results (10324-1-AP) has been validated for reactivity with human, mouse, and rat samples .

What is the evidence for synthetic lethality between MYL12A and MYL12B?

CRISPR screening data provides compelling evidence for synthetic lethality between MYL12A and MYL12B in specific cellular contexts:

• Guides targeting both MYL12A and MYL12B simultaneously showed substantially greater toxicity than guides targeting either gene individually .

• Statistical analysis confirmed a significant synergistic effect (p < 2.2 × 10^-16) when both genes were knocked out, violating the assumption of additivity in knockout effects .

• This synthetic lethality appears to be context-dependent, correlating strongly with MYL9 expression levels. The correlation between guide toxicity and MYL9 expression was substantial (Guide AB1: r = 0.534, p < 2.2 × 10^-16; Guide AB2: r = 0.377, p = 1.6 × 10^-13) .

• Cell lines with low or absent MYL9 expression showed lethal phenotypes when both MYL12A and MYL12B were targeted, suggesting that MYL9 can compensate for the loss of the other two paralogs .

This synthetic lethal interaction has important implications for understanding redundancy mechanisms within the myosin regulatory light chain family.

How do CRISPR-Cas9 off-target effects impact MYL12B research?

The search results highlight significant concerns regarding off-target effects in CRISPR-Cas9 studies of MYL12B:

• Multi-target effects: Some sgRNAs in the Avana library target both MYL12A and MYL12B, as well as pseudogenes like MYL12AP1, MYL12BP1, MYL12BP2, and MYL8P . These multi-target guides show effects that cannot be explained by simple additivity models, complicating the interpretation of gene essentiality scores.

• Single-mismatch tolerance: sgRNAs can bind to sequences with single-mismatch alignments, causing off-target effects that lead to false positive results. This issue has been documented in other gene families (e.g., SOX9/SOX10) and likely affects MYL12B studies as well .

• Biased essentiality scores: The CERES algorithm attempts to correct for multiple on-target effects by decomposing guide-specific effects as a sum of knockout effects, but this approach fails when genes exhibit non-additive interactions like synthetic lethality .

Researchers should carefully evaluate guide specificity, consider potential genetic interactions, and validate findings using orthogonal approaches when studying MYL12B using CRISPR-Cas9 technology.

What cellular phenotypes result from MYL12B knockdown versus combined MYL12A/MYL12B knockdown?

Significant differences exist between single and double knockdown phenotypes:

• Single knockdown: Guides specifically targeting either MYL12A or MYL12B alone showed LFC (log fold change) distributions centered approximately around zero, indicating minimal effect on cell viability in most cell lines .

• Double knockdown: Guides targeting both MYL12A and MYL12B resulted in substantial toxicity in a subset of cell lines, particularly those with low MYL9 expression .

• Cell structure changes: Studies with murine orthologs showed that double knockdown of Myl12a/Myl12b using siRNA caused major alterations in cell structure that were not recapitulated by isoform-specific knockdowns .

These findings suggest functional redundancy between MYL12A and MYL12B, with cells able to compensate for the loss of one paralog but not both, especially in contexts with low MYL9 expression.

How can researchers distinguish between MYL12B and its highly homologous paralogs?

Distinguishing between MYL12B and its paralogs (MYL12A and MYL9) requires careful experimental design:

• Antibody selection: Choose antibodies validated for specificity to MYL12B. The antibody referenced in the search results (10324-1-AP) has been tested for specificity to MYL12B .

• CRISPR guide design: When using CRISPR-Cas9, carefully select guides that uniquely target MYL12B. As demonstrated in the research, many guides in existing libraries co-target multiple paralogs .

• Expression analysis: Assess the expression levels of all three paralogs (MYL12A, MYL12B, and MYL9) in your experimental system to interpret phenotypes correctly.

• Rescue experiments: Perform rescue experiments with paralog-specific constructs that are resistant to the knockdown or knockout strategy being used.

• Phosphorylation-specific detection: If different phosphorylation patterns exist between the paralogs, use phospho-specific antibodies to distinguish between them.

How should researchers interpret contradictory findings in MYL12B studies?

Several factors can contribute to contradictory findings in MYL12B research:

• Genetic redundancy: The functional overlap between MYL12B, MYL12A, and MYL9 can lead to context-dependent phenotypes based on the expression levels of these paralogs .

• CRISPR guide specificity: Multi-target guides and single-mismatch tolerance can confound the interpretation of knockout phenotypes, leading to false positives and inconsistent results .

• Cell line-specific dependencies: The synthetic lethality between MYL12A and MYL12B varies with MYL9 expression levels, making findings dependent on the specific cellular context being studied .

• Analytical methods: Algorithms like CERES that assume additivity of knockout effects may produce biased essentiality scores for genes with complex genetic interactions .

When faced with contradictory findings, researchers should:

• Evaluate guide specificity and potential off-target effects

• Consider paralog expression in the experimental system

• Examine guide-level data rather than relying solely on gene-level scores

• Validate findings using orthogonal approaches

What methodological considerations are important for phosphorylation studies of MYL12B?

When studying MYL12B phosphorylation:

• Phosphorylation sites: Focus on the key regulatory sites, particularly Thr18/Ser19, which are crucial for modulating myosin II activity .

• Phospho-specific antibodies: Use antibodies that specifically recognize phosphorylated forms of MYL12B at relevant sites.

• Phosphatase inhibitors: Include appropriate phosphatase inhibitors in cell lysis buffers to preserve phosphorylation status.

• Kinase/phosphatase identification: Consider the specific kinases and phosphatases that regulate MYL12B in your experimental context.

• Functional assays: Complement phosphorylation detection with functional assays that measure myosin II activity (e.g., ATPase assays, filament assembly).

• Paralog-specific effects: Consider whether observed phosphorylation changes are specific to MYL12B or might also involve its paralogs.

How does the genetic interaction between MYL12A, MYL12B, and MYL9 affect experimental design?

The complex genetic interactions between these three paralogs necessitate careful experimental design:

• Expression profiling: Characterize the expression levels of all three paralogs in your experimental system to predict potential compensatory mechanisms.

• Single vs. double knockdowns: Consider performing both single and double knockdowns to uncover redundant functions and synthetic lethal interactions.

• MYL9 expression: Pay particular attention to MYL9 expression levels, as this appears to be a key determinant of whether MYL12A/MYL12B double knockdown will be lethal .

• Guide selection: When using CRISPR, select guides that uniquely target individual paralogs, and be aware of guides that may target multiple genes.

• Rescue experiments: Include paralog-specific rescue constructs to confirm the specificity of observed phenotypes.

• Non-additive effects: When analyzing double knockout phenotypes, be aware that effects may not be simply additive due to genetic interactions .