mical1 Antibody

What is MICAL1 and what are its key structural features?

MICAL1 is a member of the MICAL (Molecules Interacting with CasL) family of atypical multidomain flavoenzymes with diverse cellular functions. The protein contains four conserved domains: an N-terminal flavin adenine dinucleotide (FAD) binding domain, a calponin homology (CH) domain, a Lin11, Isl-1 and Mec-3 (LIM) domain, and a C-terminal coiled-coil (CC) domain . The full name of MICAL1 is microtubule associated monoxygenase, calponin and LIM domain containing 1, with a calculated molecular weight of 118 kDa, though it typically appears around 120 kDa in experimental conditions . This multi-domain structure enables MICAL1 to perform its various cellular functions, particularly in the regulation of the actin cytoskeleton.

What are the primary cellular functions of MICAL1?

MICAL1 primarily functions as a regulator of actin stress fibers and is required for normal actin organization within cells . Recent research has demonstrated that MICAL1 catalyzes redox-mediated F-actin depolymerization through the oxidation of methionine residues, including Met44 and Met47 . Beyond its role in cytoskeletal regulation, MICAL1 may be involved in apoptotic processes through its interaction with NDR (nuclear Dbf2-related) kinases . Additionally, MICAL1 participates in signaling pathways linking extracellular signals to cytoskeleton regulation via RHO GTPase family members and potentially serves as a novel means of communication between RHO and RAB GTPase signaling pathways .

What species reactivity can be expected with MICAL1 antibodies?

Common MICAL1 antibodies, such as the 68297-1-PBS monoclonal antibody, typically show reactivity with human, mouse, and rat samples . This cross-species reactivity is particularly valuable for comparative studies across different model systems. When selecting a MICAL1 antibody for your research, it's important to verify the specific species reactivity in the antibody documentation, especially if working with less common experimental models. The conservation of MICAL1 across mammalian species makes these antibodies versatile tools for various research applications.

What are the recommended applications for MICAL1 antibodies?

MICAL1 antibodies can be employed in multiple experimental techniques. For example, the 68297-1-PBS antibody has been validated for Western Blotting (WB), Immunofluorescence/Immunocytochemistry (IF/ICC), Flow Cytometry (FC) for intracellular staining, Immunoprecipitation (IP), and Indirect ELISA applications . Each application requires specific optimization for the antibody concentration and experimental conditions. For Western blotting, researchers should expect to observe a band around 120 kDa, corresponding to the observed molecular weight of MICAL1 . For immunofluorescence studies, MICAL1 typically displays a predominantly cytoplasmic localization pattern .

How can I detect MICAL1 phosphorylation in experimental systems?

Detection of phosphorylated MICAL1 can be accomplished through several approaches. Phosphorylation at specific residues, such as S817 and S960, can be assessed using phospho-specific antibodies in Western blotting or proximity ligation assays (PLA) . For PLA detection, cells can be transfected with FLAG-MICAL1, along with relevant kinases and regulators (such as CDC42 G12V and PAK1), then fixed in 4% PFA before applying antibody combinations . A pan-phospho-Ser/Thr antibody can also be used to detect general phosphorylation states when used in conjunction with immunoprecipitated MICAL1 . For increased specificity, mass spectrometry following immunoprecipitation provides a comprehensive approach to identify phosphorylation sites and quantify phosphorylation levels.

How should I design experiments to study MICAL1's role in actin dynamics?

When investigating MICAL1's impact on actin dynamics, several methodological approaches are available:

• F-actin fractionation assay: Incubate F-actin with recombinant MICAL1 (with or without activators like PAK1), then separate globular actin (G-actin) supernatant and F-actin pelleted fractions through ultracentrifugation to assess the extent of F-actin disassembly .

• Pyrene-labeled actin fluorescence assay: Monitor changes in the fluorescence of pyrene-labelled actin in real-time to assess F-actin disassembly. This involves measuring fluorescence intensity at 405 nm with excitation at 360 nm over time (e.g., every 30 seconds for 25 minutes) .

• Live-cell imaging: Transfect cells with fluorescently-tagged MICAL1 along with actin markers to visualize dynamic cytoskeletal changes in response to MICAL1 activity.

These approaches can be combined with the addition of activators (like PAK1) or inhibitors to elucidate the regulatory mechanisms of MICAL1-mediated actin reorganization.

How does the interaction between MICAL1 and PAK1 affect actin cytoskeleton regulation?

The interaction between MICAL1 and PAK1 represents a sophisticated regulatory mechanism for actin cytoskeleton dynamics. Research has shown that PAK1 binds to MICAL1 only when activated by CDC42, specifically interacting with the MO and CH domains of MICAL1 - the same domains that interact with F-actin . When active PAK1 associates with MICAL1, it significantly accelerates MICAL1-mediated F-actin disassembly. In biochemical assays, while MICAL1 alone can induce some F-actin disassembly, the addition of active PAK1 causes an even greater shift of actin from the pelleted F-actin fraction to the soluble G-actin fraction .

The mechanism involves PAK1 phosphorylation of MICAL1 at specific serine residues (S817 and S960), which appears to relieve MICAL1's autoinhibited state . This phosphorylation subsequently enhances MICAL1's catalytic activity toward F-actin and promotes additional protein interactions, suggesting a model where PAK1 activation of MICAL1 creates a signaling pathway linking extracellular signals through RHO GTPases to cytoskeletal reorganization .

What protein-protein interactions are affected by MICAL1 phosphorylation?

MICAL1 phosphorylation by PAK1 significantly alters its protein interaction network. Mass spectrometry analysis of co-immunoprecipitated proteins reveals that phosphorylated MICAL1 associates with a larger number of proteins (473 increased versus 216 decreased) compared to non-phosphorylated MICAL1 . The magnitude of difference in binding is also greater for proteins showing increased association.

Key proteins showing enhanced binding to phosphorylated MICAL1 include:

• PAK1 and CDC42 - confirming the stability of this regulatory complex

• RAB7A and RAB10 - previously identified MICAL1 interacting proteins

• YWHAE (14-3-3ε) - a phosphoserine-binding protein

These interactions suggest that PAK1-mediated phosphorylation of MICAL1 may create a conformational change that not only enhances its enzymatic activity but also exposes binding sites for additional regulatory proteins . The increased association with RAB GTPases particularly suggests a novel link between RHO and RAB GTPase signaling pathways, potentially connecting cytoskeletal regulation with membrane trafficking processes .

How does MICAL1 catalytic activity compare across different experimental conditions?

The catalytic activity of MICAL1 in F-actin disassembly varies significantly under different experimental conditions. Comparative data from pyrene-labeled actin fluorescence assays demonstrates these differences:

The synergistic effect of active PAK1 and MICAL1 suggests a regulatory mechanism where PAK1 increases MICAL1's inherent activity . This increased activity correlates with PAK1-mediated phosphorylation of MICAL1 at S817 and S960, which can be inhibited by the group I PAK inhibitor FRAX1036 . These observations provide a framework for understanding how post-translational modifications can fine-tune MICAL1's enzymatic functions in cellular contexts.

What is the recommended protocol for immunoprecipitation of MICAL1?

For successful immunoprecipitation of MICAL1, the following protocol is recommended:

• Cell preparation: Culture cells (e.g., HEK293T) in appropriate media supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics .

• Transfection: If studying exogenous MICAL1, transfect cells with FLAG-tagged MICAL1 constructs along with any relevant interacting proteins (e.g., PAK1, CDC42) .

• Cell lysis: Lyse cells in a buffer containing protease inhibitors (such as cOmplete™ Protease Inhibitor Cocktail) and phosphatase inhibitors if studying phosphorylation states .

• Immunoprecipitation: Incubate cell lysates with anti-FLAG antibody (for tagged MICAL1) or specific MICAL1 antibodies conjugated to appropriate beads (e.g., Protein G-Sepharose) .

• Washing: Perform multiple washes with lysis buffer to remove non-specific binding proteins.

• Elution: Elute bound proteins using SDS sample buffer for Western blotting analysis or perform on-bead digestion for mass spectrometry applications .

For phosphorylation studies, additional steps may include in vitro kinase assays with recombinant active PAK1 and ATP before elution .

What controls should be included when studying MICAL1 phosphorylation?

When investigating MICAL1 phosphorylation, several critical controls should be included:

• Kinase-dead mutants: Include catalytically inactive kinase mutants (e.g., PAK1 K299R) to demonstrate phosphorylation specificity .

• Phosphorylation site mutants: Generate MICAL1 constructs with alanine substitutions at putative phosphorylation sites (e.g., S817A, S960A) to confirm the specific residues targeted by kinases .

• Kinase inhibitors: Employ specific inhibitors (such as FRAX1036 for group I PAKs) to validate kinase dependency of the observed phosphorylation .

• Phosphatase treatment: Include samples treated with phosphatases to confirm antibody phospho-specificity.

• Total protein controls: Always assess total MICAL1 levels alongside phosphorylated forms to normalize phosphorylation signals.

These controls help distinguish specific kinase-mediated phosphorylation from background or non-specific signals and provide confidence in the interpretation of results.

How can proximity ligation assays be optimized for detecting phosphorylated MICAL1?

Proximity Ligation Assay (PLA) provides a powerful approach for detecting and localizing phosphorylated MICAL1 in situ. To optimize this technique:

• Cell preparation: Transfect cells (e.g., U2OS) with FLAG-MICAL1, relevant kinases (PAK1WT), and upstream activators (CDC42 G12V), then fix in 4% PFA .

• Antibody selection: Use a primary antibody combination consisting of:

  • Mouse anti-FLAG antibody to detect total MICAL1

  • Rabbit phospho-specific antibodies (anti-pS817 or anti-pS960) to detect phosphorylated residues

• Controls: Include parallel samples with kinase-dead mutants (PAK1KD) to demonstrate phosphorylation specificity .

• Signal detection: Employ a Duolink® PLA kit or similar system with species-specific secondary antibodies and amplification reagents.

• Imaging: Use high-resolution confocal microscopy for optimal visualization of PLA signals.

For accurate interpretation, compare PLA signals for phosphorylated MICAL1 to those for total MICAL1 expression detected using pairs of mouse and rabbit anti-FLAG antibodies . This approach allows for the specific detection of phosphorylated MICAL1 in its native cellular context.

Why might I observe differences between calculated and observed molecular weights for MICAL1?

The discrepancy between calculated (118 kDa) and observed (120 kDa) molecular weights of MICAL1 in Western blotting is a common phenomenon that can be attributed to several factors :

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can increase the apparent molecular weight by altering protein mobility in SDS-PAGE.

• Protein structure: The presence of structured domains in MICAL1 might affect SDS binding and alter migration patterns.

• Gel concentration and running conditions: The percentage of acrylamide and running buffer composition can affect mobility.

• Protein standards calibration: Variations in molecular weight standards can lead to differences in calculated weights.

If consistent observation of MICAL1 at 120 kDa is critical for your research, consider using known MICAL1-expressing cell lysates as positive controls to establish the expected migration pattern for your specific experimental conditions .

How can I improve specificity when studying MICAL1 in complex protein mixtures?

Improving specificity for MICAL1 detection in complex samples requires multiple approaches:

• Antibody validation: Confirm antibody specificity using knockout/knockdown controls or competing peptides specific to the antibody epitope.

• Immunoprecipitation: Enrich for MICAL1 before analysis to reduce background and non-specific signals .

• Multiple antibody approach: Utilize different antibodies recognizing distinct epitopes on MICAL1 to confirm observations.

• Domain-specific antibodies: For studies focusing on particular functions, consider antibodies that specifically recognize functional domains of MICAL1.

• Mass spectrometry verification: Confirm Western blot or immunofluorescence results with mass spectrometry identification of MICAL1-specific peptides .

For phosphorylation studies, the combination of phospho-specific antibodies with pan-phospho-Ser/Thr antibodies following immunoprecipitation provides an effective strategy to enhance detection specificity .

What are the storage and handling recommendations to maintain MICAL1 antibody activity?

To maintain optimal activity of MICAL1 antibodies:

• Storage temperature: Store antibodies at -80°C for long-term preservation .

• Aliquoting: Upon receipt, divide the antibody into small working aliquots to avoid repeated freeze-thaw cycles.

• Buffer conditions: Maintain antibodies in appropriate buffers (e.g., PBS) with preservatives if needed .

• Working dilutions: Prepare fresh working dilutions for each experiment rather than storing diluted antibody.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination.

• Transport: Transport antibodies on ice and minimize exposure to room temperature.

Following these recommendations will help maintain antibody specificity and sensitivity, ensuring reliable and reproducible results across experiments.

How might MICAL1 function in the cross-talk between RHO and RAB GTPase signaling pathways?

The discovery that phosphorylated MICAL1 shows increased binding to both RAB GTPases (RAB7A, RAB10) and components of the RHO GTPase pathway (CDC42, PAK1) suggests a potential role for MICAL1 as a molecular bridge between these signaling networks . Future research could explore:

• The spatial and temporal dynamics of MICAL1-mediated interactions between RHO and RAB GTPases in cellular processes like vesicle trafficking and cytoskeletal reorganization.

• Whether MICAL1 phosphorylation serves as a molecular switch to coordinate transitions between RHO-mediated cytoskeletal dynamics and RAB-controlled membrane trafficking events.

• The potential role of MICAL1 in specialized cellular contexts where coordinated membrane and cytoskeletal remodeling occurs, such as cell migration, phagocytosis, or neuronal development.

• Development of biosensors to visualize MICAL1 activation states in real-time during interactions with different GTPase pathways.

These investigations could reveal fundamental mechanisms of cellular integration between cytoskeletal regulation and membrane dynamics .

What are the implications of MICAL1 in disease processes and potential therapeutic applications?

Given MICAL1's role in cytoskeletal dynamics and its connections to signaling pathways, future research might explore its involvement in:

• Cancer progression: Investigate MICAL1's contribution to cancer cell migration, invasion, and metastasis through its effects on actin reorganization and potential connections to oncogenic signaling pathways.

• Neurodegenerative diseases: Explore MICAL1's potential role in axonal degeneration or synaptic dysfunction, given the importance of actin dynamics in neuronal health.

• Inflammatory disorders: Study MICAL1's function in immune cell migration and activation in the context of inflammatory responses.

• Therapeutic targeting: Develop small molecule inhibitors or activators of MICAL1 that could modulate its activity in disease contexts.

• Biomarker potential: Assess whether MICAL1 expression or phosphorylation status correlates with disease progression or treatment response in various pathological conditions.

These directions could open new avenues for understanding disease mechanisms and identifying novel therapeutic strategies targeting cytoskeletal regulation.