DRP1A Antibody refers to immunological reagents designed to detect Dynamin-Related Protein 1A (DRP1A), a plant-specific dynamin GTPase in Arabidopsis thaliana. DRP1A is critical for cell plate formation, endocytosis, and immune signaling, particularly in responses to bacterial pathogens like Pseudomonas syringae. While DRP1A is distinct from mammalian DRP1 (Dynamin-1-like protein), antibodies for DRP1 are often cross-referenced in literature due to shared functional domains.
No commercial antibodies for DRP1A are listed in available databases.
Plant studies rely on custom antibodies or cross-reactivity assumptions.
Cytokinesis: DRP1A is essential for cell plate formation, as evidenced by defective cytokinesis in drp1a mutants .
Cell Expansion: Mutants show stunted roots/cotyledons and defective PIN protein localization, affecting auxin transport .
FLS2 Regulation: DRP1A mediates ligand-induced endocytosis of FLS2, balancing immune activation and downregulation .
Pathogen Response:
Double mutants (drp1a drp2b) exhibit seedling lethality, hyperaccumulation of FLS2 in PM, and blocked flg22-induced endocytosis .
Lipid Environment Modulation: DRP1A and DRP2B may regulate sterol-rich PM domains to maintain signaling competency .
Antibody Specificity:
Current antibodies target mammalian DRP1, not plant DRP1A.
Need: Develop plant-specific antibodies for precise localization studies.
Mechanistic Insights:
How DRP1A and DRP2B coordinate endocytosis and membrane remodeling?
Role of lipid domains in FLS2 signaling.
Agricultural Applications:
Engineering DRP1A/DRP2B activity to enhance pathogen resistance.
DRP1A (also known as ADL1, ADL1A, AG68, DL1, DYNAMIN-LIKE PROTEIN, DYNAMIN-RELATED PROTEIN 1A, RADIAL SWELLING 9, or RSW9) is a dynamin-like protein related to phragmoplastin in plants. This protein functions in multiple essential cellular processes including embryogenesis, cell plate formation during cytokinesis, trichome branching, and vascular patterning in coordination with VAN3 and GNOM . Significantly, DRP1A participates with DRP2B in clathrin-coated vesicle formation during endocytosis, making it a key component of the plant endocytic machinery . Research has demonstrated that DRP1A plays critical roles in plant immunity responses, particularly by regulating the PM-associated levels of the flagellin receptor FLS2, which is necessary for plants to initiate distinct immune signaling pathways .
The preferred methodology for DRP1A detection involves immunoblotting using specific anti-DRP1A antibodies. For optimal results, researchers should:
Extract total proteins from plant tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitors
Separate proteins via SDS-PAGE (10-12% gels work well for the ~68 kDa DRP1A)
Transfer to PVDF or nitrocellulose membranes
Block with 5% non-fat milk in TBST
Incubate with anti-DRP1A primary antibody (typically at 1:1000-1:2000 dilution)
Visualize using appropriate secondary antibody and detection system
For immunocytochemical analyses, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 provides good results for subcellular localization studies . When probing for DRP1A in various plant species, researchers should consider cross-reactivity profiles, as the commercially available antibodies show reactivity with DRP1A from multiple plant species including Arabidopsis thaliana, Brassica species, Solanum species, and various cereal crops .
When conducting DRP1A antibody-based experiments, implementing appropriate controls is crucial for result validation:
Research has validated the specificity of anti-DRP1A antibodies by demonstrating absence of signal in drp1a mutants, while maintaining detection in wild-type controls . This type of genetic validation provides the strongest evidence for antibody specificity in plant research.
DRP1A plays significant roles in plant immunity, and antibody-based approaches offer powerful insights into these functions. To investigate DRP1A's role in plant immunity:
Compare DRP1A protein levels in plants before and after pathogen exposure using quantitative immunoblotting
Conduct co-immunoprecipitation with anti-DRP1A antibody to identify immune-related interaction partners
Perform immunofluorescence microscopy to track DRP1A relocalization during immune responses
Use proximity ligation assays with anti-DRP1A and antibodies against known immune components
Research has revealed that DRP1A contributes positively to pattern-triggered immunity (PTI) against bacterial pathogens like Pseudomonas syringae pv. tomato DC3000 . Immunoblotting analysis has shown that while drp1a mutants maintain normal steady-state levels of the immune receptor FLS2, they exhibit defects in ligand-induced receptor endocytosis . These findings suggest DRP1A regulates receptor dynamics rather than baseline expression, highlighting the value of temporal studies using anti-DRP1A antibodies.
DRP1A and DRP2B demonstrate functional overlap in endocytosis and immune signaling. To investigate their interactions:
Perform sequential immunoprecipitation with both anti-DRP1A and anti-DRP2B antibodies
Conduct immunofluorescence co-localization studies using antibodies against both proteins
Apply proximity-dependent biotinylation (BioID) coupled with immunodetection
Analyze protein dynamics in drp1a, drp2b, and drp1a drp2b double mutants
Research has demonstrated that DRP1A and DRP2B exhibit subcellular co-localization and direct intermolecular interactions . Both proteins contribute to clathrin-mediated endocytosis, with drp1a mutants showing more severe defects in ligand-induced endocytosis of the FLS2 receptor compared to drp2b mutants . Comparative immunoblotting of single and double mutants has revealed that DRP1A protein is undetectable in drp1a single and drp1a drp2b double mutants, providing important genetic tools for studying these interactions .
DRP1A function is regulated by various post-translational modifications. To differentiate between these modifications:
Use phospho-specific antibodies that recognize specific phosphorylation sites
Employ Phos-tag™ SDS-PAGE followed by anti-DRP1A immunoblotting
Conduct 2D gel electrophoresis (separating by isoelectric point and molecular weight) with anti-DRP1A detection
Perform mass spectrometry analysis following DRP1A immunoprecipitation
While specific information about DRP1A phosphorylation in plants is limited in the provided search results, studies on mammalian Drp1 have demonstrated the importance of phosphorylation at specific sites (e.g., S616) in regulating its function . Similar phosphorylation events likely regulate plant DRP1A activity, particularly during developmental transitions or stress responses. Researchers investigating these modifications should consider combining immunoprecipitation using anti-DRP1A antibodies with phosphoproteomic analysis to identify regulatory sites.
Inconsistent DRP1A detection can result from several technical and biological factors:
Factor | Explanation | Solution |
---|---|---|
Variable expression | DRP1A expression differs between tissues and developmental stages | Include positive control samples with known high DRP1A expression |
Protein extraction issues | Different tissues require optimized extraction methods | Test multiple extraction buffers; include reducing agents and protease inhibitors |
Fixation effects | Overfixation may mask epitopes in immunohistochemistry | Optimize fixation time; try epitope retrieval methods |
Post-translational modifications | Modifications may alter antibody recognition | Use multiple antibodies targeting different epitopes |
Research has revealed that DRP1A functions in various developmental processes, including embryogenesis, cell plate formation, and trichome branching . This suggests tissue-specific regulation that may affect detection. When comparing DRP1A levels across tissues, researchers should normalize to appropriate loading controls and consider using multiple antibodies targeting different epitopes to ensure comprehensive detection.
Optimizing DRP1A immunoprecipitation requires careful consideration of several parameters:
Buffer composition:
For membrane-associated interactions, include 0.5-1% digitonin or 0.5% NP-40
For stronger interactions, RIPA buffer may be suitable
Always include protease and phosphatase inhibitors
Antibody coupling:
Direct coupling to beads (using BS3 or DMP) prevents antibody contamination in eluates
For weaker interactions, gentle cross-linking approaches are preferable
Incubation conditions:
Overnight incubation at 4°C with gentle rotation
Pre-clear lysates with protein A/G beads to reduce non-specific binding
Validation approaches:
Confirm IP efficiency by immunoblotting 5-10% of input, unbound, and elution fractions
Include IgG control and, when available, immunoprecipitation from drp1a mutant tissue
Based on research showing DRP1A interactions with DRP2B in endocytic processes , immunoprecipitation protocols should be designed to preserve membrane-associated protein complexes. The subcellular co-localization and intermolecular interaction between these proteins suggest that membrane solubilization conditions are critical for successful co-immunoprecipitation experiments.
Researchers sometimes encounter discrepancies between protein levels detected by antibodies and mRNA expression. To resolve such contradictions:
Validate antibody specificity:
Compare detection in wild-type vs. drp1a mutants
Perform peptide competition assays
Test multiple antibodies targeting different epitopes
Assess post-transcriptional regulation:
Measure protein half-life using cycloheximide chase experiments
Investigate miRNA-mediated regulation
Examine polysome association of DRP1A transcripts
Consider technical limitations:
Different sensitivities between RT-qPCR and immunoblotting
Non-linear relationship between transcript and protein
Tissue-specific translation efficiency
Research has demonstrated that while drp1a mutants show highly reduced DRP1A mRNA levels, protein detection using antibodies provides the definitive proof of null mutation status . This highlights the complementary nature of transcript and protein analysis. In some experimental systems, researchers have observed no differences in steady-state FLS2 mRNA or protein levels in drp1a mutants compared to wild-type plants, even though functional differences exist in receptor dynamics , emphasizing the importance of analyzing both static levels and dynamic processes.
DRP1A antibodies enable comparative studies across diverse plant species:
Cross-species immunoblotting:
Assess conservation and divergence in protein size and abundance
Compare expression patterns across developmental stages
Evaluate responses to environmental stressors
Immunolocalization studies:
Compare subcellular localization patterns
Assess recruitment to endocytic sites
Evaluate co-localization with conserved endocytic markers
Experimental considerations:
Select antibodies with broad cross-reactivity profiles
Adjust protein extraction protocols for species-specific tissue composition
Validate specificity in each species through appropriate controls
The available anti-DRP1A antibodies show reactivity with a wide range of plant species including Arabidopsis thaliana, Brassica species, Spinacia oleracea, Cucumis sativus, Vitis vinifera, Solanum species, Populus trichocarpa, Medicago truncatula, Glycine max, and various cereal crops including Zea mays, Oryza sativa, Panicum virgatum, Setaria viridis, Sorghum bicolor, Triticum aestivum, and Hordeum vulgare . This broad reactivity stems from conservation of the immunizing peptide sequence, with the synthetic peptide used for immunization being 93% (14/15 amino acids) homologous with the sequence in the related protein DL1B (AT3G61760) .
While sharing functional similarities, plant DRP1A and mammalian Drp1 exhibit important differences:
Feature | Plant DRP1A | Mammalian Drp1 | Experimental Implications |
---|---|---|---|
Cellular processes | Cell plate formation, endocytosis, immunity | Mitochondrial fission, peroxisomal division | Different subcellular markers needed |
Disease associations | Plant development, pathogen resistance | Neurodegenerative disorders, cardiovascular disease, cancer | Different phenotypic readouts |
Post-translational regulation | Less characterized | Well-characterized phosphorylation (e.g., S616) | Different modification-specific antibodies required |
Interaction partners | DRP2B, clathrin, endocytic machinery | Mitochondrial receptors (e.g., Mff, Fis1) | Different co-immunoprecipitation targets |
Research on mammalian Drp1 has revealed its involvement in neurodegenerative diseases (Alzheimer's, Huntington's, Parkinson's), cardiovascular diseases, and various cancers . In contrast, plant DRP1A functions primarily in developmental processes and immunity. When designing experiments using antibodies that might cross-react between plant and animal systems, researchers should carefully validate specificity and be aware that regulatory mechanisms likely differ significantly between kingdoms.
Nanobodies offer exciting new approaches for studying DRP1A dynamics:
Advantages over traditional antibodies:
Smaller size (approximately 15 kDa vs. 150 kDa for IgG)
Greater epitope accessibility in complex structures
Potential for live-cell imaging when fused to fluorescent proteins
Greater stability under various experimental conditions
Potential applications:
Live-cell tracking of DRP1A dynamics during endocytosis
Super-resolution microscopy with minimal linkage error
Manipulation of DRP1A function through targeted nanobody binding
Proximity-dependent labeling with nanobody-enzyme fusions
Recent research has developed nanobodies as versatile tools for proteomics, advanced microscopy, and live cell imaging of mammalian Drp1 . Similar approaches could be applied to plant DRP1A, enabling unprecedented insights into its dynamic behavior during cell division, endocytosis, and immune responses. When combining nanobody and traditional antibody approaches, researchers should consider epitope competition and validate that nanobody binding doesn't interfere with DRP1A function.
Integrating DRP1A antibody detection with single-cell approaches presents exciting opportunities:
Single-cell protein profiling:
Combine flow cytometry with intracellular DRP1A staining
Apply mass cytometry (CyTOF) with metal-conjugated anti-DRP1A antibodies
Implement microfluidic platforms for single-cell Western blotting
Spatial analysis:
Employ imaging mass cytometry to map DRP1A distribution in tissues
Use multiplexed immunofluorescence to correlate DRP1A with other proteins
Apply proximity ligation assays to detect specific interactions in situ
Experimental considerations:
Optimize cell isolation protocols to maintain protein integrity
Validate antibody specificity in single-cell formats
Develop appropriate normalization standards for quantitative analysis
While single-cell approaches are still emerging in plant science, DRP1A's involvement in fundamental cellular processes makes it an ideal target for techniques that can resolve cell-to-cell variability. The observation that DRP1A functions in diverse processes including embryogenesis, cell plate formation, trichome branching, and immunity suggests that its activity may vary substantially between cell types, making single-cell analysis particularly informative.