OSCA1 Antibody

What is the OSCA/TMEM63 protein family and how is OSCA1 classified within it?

The OSCA/TMEM63 family represents one of the largest families of mechanically activated ion channels identified to date, with conservation across eukaryotes. In plants, the OSCA family consists of 15 isoforms grouped into 4 different phylogenetic clades . OSCA1 belongs to the first clade and includes several members such as OSCA1.1, OSCA1.2, and OSCA1.3, each with distinct functional characteristics and expression patterns. This family is particularly significant as it forms pore-forming ion channels that respond to mechanical stimuli and hyperosmotic stress . When designing experiments with OSCA1 antibodies, researchers should consider the specific isoform they are targeting, as cross-reactivity between closely related family members may occur.

What are the key structural features of OSCA1 proteins that antibodies might target?

OSCA1 proteins are plasma membrane-localized ion channels that function as dimers, with each subunit containing a pore . Key structural features include:

• Multiple transmembrane domains

• Cytoplasmic loops (particularly the first cytoplasmic loop containing important regulatory sites)

• N- and C-terminal domains

• Phosphorylation sites (notably S54 in OSCA1.3)

The cryo-EM structures of OSCA1.1 and OSCA1.2 have revealed a pore within each subunit of a dimeric channel . When designing or selecting antibodies, researchers should consider targeting unique epitopes that distinguish between OSCA isoforms, particularly in the more variable cytoplasmic regions, while avoiding the highly conserved transmembrane domains if isoform specificity is desired.

What are the recommended methods for validating OSCA1 antibody specificity?

When validating OSCA1 antibody specificity, researchers should employ multiple complementary approaches:

• Western blotting with positive and negative controls: Use tissue from wild-type plants and osca1 knockout mutants (e.g., osca1.3/1.7 double mutants as described in the literature) .

• Immunoprecipitation followed by mass spectrometry: This can confirm the antibody is pulling down the correct protein and can identify potential cross-reactive proteins.

• Immunolocalization studies: Compare antibody staining patterns with the known subcellular localization of OSCA1 proteins (plasma membrane) .

• Pre-absorption controls: Pre-incubate antibodies with purified antigen to demonstrate specificity of the signal.

• Heterologous expression systems: Test antibodies against OSCA1 proteins expressed in systems like HEK293F cells, which have been successfully used for OSCA1.2 expression and purification .

What electrophysiological techniques are most appropriate for functional studies of OSCA1 when validating antibody effects?

Several electrophysiological approaches have proven effective for studying OSCA1 function:

• Whole-cell patch clamp with mechanical stimulation: This technique has been used to record mechanically activated currents from cells expressing OSCA1.1 or OSCA1.2. Mechanical stimulation can be achieved using a fire-polished glass pipette (tip diameter 3-4 μm) positioned at an 80° angle relative to the cell .

• Cell-attached or excised patch clamp with pressure stimulation: This enables recording of stretch-activated currents by applying negative or positive pressure pulses through the recording electrode using a pressure clamp device .

• Single-channel recordings: These have revealed that OSCA1.1 and OSCA1.2 have single-channel conductances of 184 ± 4 pS and 122 ± 3 pS, respectively, with subconductance states that are half the amplitude of the full open state .

When assessing antibody effects on channel function, researchers should consider adding antibodies to the bath solution (for accessible extracellular epitopes) or including them in the patch pipette solution (for intracellular epitopes) while monitoring changes in channel properties.

How is OSCA1.3 regulated by phosphorylation and what techniques can detect these modifications?

OSCA1.3 is regulated by phosphorylation via the immune-related kinase BIK1 (Botrytis-induced kinase 1). Key findings include:

• Phosphorylation sites: The primary site is S54 in the first cytoplasmic loop of OSCA1.3, with S49 as a secondary site. These sites are within a conserved SxxL motif .

• Detection methods:

  • In vitro kinase assays: Radioactive kinase assays using purified proteins have demonstrated that BIK1 specifically phosphorylates OSCA1.3 at S54 .

  • Selected-reaction monitoring (SRM) assays: These have confirmed flg22-induced, BIK1-dependent phosphorylation of S54 in vivo .

  • Co-immunoprecipitation experiments: These have shown that BIK1 physically interacts with OSCA1.3 .

• Functional significance: Phosphorylation at S54 is critical for OSCA1.3 function in immunity, as demonstrated by the inability of the OSCA1.3-S54A mutant to restore flg22-induced stomatal closure in osca1.3/1.7 mutant plants .

When using antibodies to study OSCA1 phosphorylation, researchers should consider developing phospho-specific antibodies targeting key regulatory sites like S54 in OSCA1.3.

What advanced approaches can be used to study the interaction between OSCA1 and regulatory proteins?

Several sophisticated techniques can be employed to study OSCA1 interactions with regulatory proteins:

• Proximity labeling methods: Techniques like BioID or TurboID, where OSCA1 is fused to a biotin ligase to identify proteins in close proximity in living cells.

• FRET/BRET analysis: To visualize protein-protein interactions in real-time in living cells.

• Co-immunoprecipitation with antibodies: As demonstrated for BIK1-HA and OSCA1.3-GFP , this approach can identify interacting partners.

• Split-fluorescent protein complementation: For visualizing protein interactions in planta.

• Surface plasmon resonance or microscale thermophoresis: For quantitative measurement of binding affinities between purified OSCA1 and partner proteins.

• Cryo-EM structural studies: The structures of OSCA1.1 and OSCA1.2 have been determined , and similar approaches could be used to study regulatory protein complexes.

When using antibodies in these interaction studies, researchers should verify that the antibody epitope does not overlap with or disrupt interaction interfaces.

What methods are recommended for measuring OSCA1-mediated calcium influx in plant cells?

Several approaches have been effectively used to measure OSCA1-mediated calcium influx:

When using antibodies to study OSCA1-mediated calcium signaling, researchers can assess whether antibody binding affects calcium influx in these assay systems.

How can researchers distinguish between OSCA1-mediated calcium influx and other calcium signaling pathways?

Distinguishing OSCA1-specific calcium signaling from other pathways requires strategic experimental design:

• Genetic approaches:

  • Use of osca1 knockout mutants (e.g., osca1.3/1.7)

  • Complementation studies with wild-type OSCA1 versus mutant versions (e.g., OSCA1.3-S54A)

  • Comparison with other calcium channel mutants

• Pharmacological approaches:

  • Channel blockers such as gadolinium (Gd³⁺), which has been used in OSCA studies

  • Specific inhibitors of other calcium channels

• Stimulus specificity:

  • OSCA1.3/1.7 are specifically required for flg22-induced but not ABA-induced stomatal closure , indicating pathway specificity

  • Mechanical stimulation for OSCA1.1/1.2 versus osmotic shock or immune elicitors for other family members

• Temporal resolution: Monitoring the kinetics of calcium responses with high-resolution imaging

When using antibodies to dissect calcium signaling pathways, researchers might apply specific antibodies to different OSCA isoforms and assess their effects on distinct calcium responses.

What are the established roles of OSCA1 proteins in plant immune responses?

OSCA1 proteins, particularly OSCA1.3, play crucial roles in plant immunity:

• Guard cell immunity: OSCA1.3/1.7 are required for flg22-induced stomatal closure, a critical early defense response that prevents pathogen entry .

• Calcium signaling: OSCA1.3 mediates calcium influx in guard cells upon perception of the bacterial PAMP flg22, with osca1.3/1.7 mutants showing reduced calcium spiking compared to wild-type plants .

• Disease resistance: osca1.3/1.7 mutant plants show increased susceptibility to the hypovirulent Pseudomonas syringae pv tomato DC3000 COR- strain, comparable to the immune-deficient mutant bak1-5 .

• Pathway specificity: OSCA1.3/1.7 function specifically in immune-related calcium signaling, as they are not required for ABA-induced stomatal closure .

• Regulation by immune signaling: OSCA1.3 is phosphorylated by the immune-related kinase BIK1 at residue S54, and this phosphorylation is essential for its function in immunity .

For antibody-based studies of OSCA1 in immunity, researchers should consider comparing immune responses in the presence of antibodies that target different OSCA1 epitopes, potentially revealing which domains are critical for immune function.

What advanced experimental setups can be used to study OSCA1 function in mechanosensing and osmosensing?

Sophisticated experimental approaches for investigating OSCA1 mechanosensing and osmosensing include:

• Mechanical stimulation techniques:

  • Membrane indentation: Using a glass probe controlled by a piezo-electric crystal microstage to apply defined mechanical stimuli and measure activation thresholds (6.3-8.6 μm for OSCA1.1/1.2) .

  • Membrane stretch: Applying negative or positive pressure through recording electrodes to induce stretch-activated currents, determining pressure for half-maximal activation (P50) (-54.5 to -58.5 mmHg for OSCA1.1/1.2) .

• Osmotic stress assays:

  • Controlled osmotic shifts: Switching between iso-osmotic (300 mmol.kg⁻¹) and hyper-osmotic (620 mmol.kg⁻¹) solutions while recording channel activity .

  • Ramp protocols: Voltage ramps from -100 mV to +100 mV to characterize channel responses to osmotic changes .

• Single-molecule techniques:

  • Atomic force microscopy to measure force-dependent conformational changes

  • Single-molecule FRET to monitor channel conformational states

• Computational approaches:

  • Molecular dynamics simulations of OSCA1 proteins in membranes under tension

Antibodies could be used in these setups to test whether binding to specific domains affects mechanosensitivity or osmosensitivity, potentially revealing the sensor domains of these channels.

What are the major challenges in generating specific antibodies against different OSCA1 isoforms?

Developing isoform-specific antibodies for OSCA1 proteins presents several challenges:

• Sequence homology: The high sequence similarity between OSCA isoforms (particularly within the same clade) makes it difficult to identify unique epitopes for antibody generation.

• Membrane protein structure: As integral membrane proteins, OSCA1 proteins have limited exposed regions for antibody targeting, primarily the cytoplasmic loops and termini.

• Post-translational modifications: Modifications like phosphorylation (e.g., at S54 in OSCA1.3) may affect antibody binding and should be considered when selecting epitopes.

• Expression and purification: Obtaining sufficient quantities of purified OSCA1 proteins for antibody production and validation is challenging, though successful expression in HEK293F cells has been reported .

• Conformational epitopes: The native conformation of OSCA1 proteins in the membrane may present epitopes that are lost during denaturation for techniques like Western blotting.

To address these challenges, researchers might consider:

• Targeting the more variable N- or C-terminal regions or cytoplasmic loops

• Developing monoclonal antibodies with extensive screening for specificity

• Creating recombinant antibody fragments (Fabs, scFvs) from phage display libraries

• Using synthetic peptide antigens corresponding to unique regions of specific OSCA1 isoforms

How can researchers troubleshoot non-specific binding or low sensitivity issues with OSCA1 antibodies?

When encountering problems with OSCA1 antibodies, consider these troubleshooting approaches:

• For non-specific binding:

  • Increase blocking stringency (e.g., 5% BSA or milk instead of 3%)

  • Optimize antibody dilution through titration experiments

  • Include competing peptides corresponding to shared epitopes of non-target OSCA isoforms

  • Use knockout tissue (e.g., osca1.3/1.7) to identify non-specific bands

  • Increase washing stringency (higher salt concentration, longer wash times)

  • Pre-absorb antibodies with tissue from knockout plants

• For low sensitivity:

  • Optimize protein extraction methods for membrane proteins (consider detergent choice)

  • Try signal amplification methods (e.g., biotin-streptavidin systems)

  • Enrich target proteins through immunoprecipitation before detection

  • Test different epitope retrieval methods for fixed tissues

  • Increase antibody incubation time or temperature

  • Try different detection systems (fluorescent vs. chemiluminescent)

• For both issues:

  • Compare polyclonal vs. monoclonal antibodies

  • Validate with epitope-tagged versions of the protein (e.g., OSCA1.3-GFP)

  • Consider the effect of phosphorylation status on antibody binding, particularly for OSCA1.3 where S54 phosphorylation is important

What emerging technologies might enhance OSCA1 antibody-based research?

Several cutting-edge technologies could advance OSCA1 antibody research:

• Nanobodies/single-domain antibodies: These smaller antibody fragments offer advantages for targeting less accessible epitopes in membrane proteins like OSCA1.

• Proximity-dependent labeling: Combining antibodies with enzymes like APEX2 or TurboID for identifying proteins in the vicinity of OSCA1 in native contexts.

• Super-resolution microscopy: Techniques like STORM, PALM, or STED combined with highly specific antibodies could reveal nanoscale organization of OSCA1 channels in the membrane.

• Cryo-electron tomography: For visualizing OSCA1 complexes in their native membrane environment at near-atomic resolution.

• Optogenetic tools combined with antibodies: For spatiotemporal control of OSCA1 function and simultaneous detection.

• Single-cell proteomics: To analyze OSCA1 expression and modifications in specific cell types like guard cells where OSCA1.3 is preferentially expressed .

• Intrabodies: Engineered antibodies that function inside living cells to monitor or modulate OSCA1 function in real-time.

How might OSCA1 antibodies be used to investigate unresolved questions about channel activation mechanisms?

OSCA1 antibodies could help resolve several key mechanistic questions:

• Force sensing domains:

  • Using antibodies targeting specific domains to block or alter mechanosensitivity

  • Comparing effects on mechanical stimulation versus osmotic activation pathways

  • Testing whether antibody binding to putative sensor domains alters activation thresholds (6.3-8.6 μm for indentation, -54.5 to -58.5 mmHg for stretch)

• Channel gating mechanisms:

  • Probing the significance of subconductance states observed in OSCA1.1 and OSCA1.2

  • Investigating whether antibodies that bind near the dimeric interface affect the concerted gating suggested by the single sub-conductance state

  • Exploring how phosphorylation (e.g., at S54 in OSCA1.3) changes channel gating properties

• Regulatory protein interactions:

  • Using antibodies to disrupt or detect specific interactions, such as between BIK1 and OSCA1.3

  • Investigating whether different stimuli recruit different regulatory partners to OSCA1 channels

• Structure-function relationships:

  • Correlating antibody epitope mapping with functional domains identified in cryo-EM structures

  • Exploring whether antibodies can distinguish between different conformational states of the channel

These approaches could help elucidate the molecular mechanisms by which OSCA1 channels sense and respond to mechanical forces and osmotic stress, as well as how these mechanisms are regulated during plant immunity and other stress responses.