DRP5A Antibody

What is DRP5A and what is its cellular function in plants?

DRP5A is a dynamin-related protein that functions in plant cytokinesis rather than chloroplast division. Studies with DRP5A-GFP and anti-DRP5A antibodies have shown that DRP5A localizes to the cell plate during cytokinesis, with expression limited to dividing cells in meristematic tissues. Immunofluorescence microscopy has revealed that DRP5A exhibits a cell cycle-dependent localization pattern, with speckles appearing around the nucleus during prophase and eventually accumulating at the cell plate during late telophase . M phase-specific accumulation of DRP5A mRNA has been detected through cell cycle synchronization in Arabidopsis thaliana cultured cells, indicating that DRP5A expression is tightly regulated during the cell cycle .

How does DRP5A differ from other dynamin-related proteins in plants?

DRP5A belongs to a family of dynamin-related proteins that includes DRP5B, its closest relative. Despite their structural similarity, these proteins have distinct functions:

Unlike DRP5B, which localizes to both chloroplasts and peroxisomes and is involved in their division, DRP5A is specifically involved in cytokinesis . The phylogenetic distribution of DRP5A (found in Viridiplantae, Amoebozoa, and Heterolobosea) suggests an ancient role in cell division mechanisms that has been conserved in multiple evolutionary lineages .

What approaches are recommended for generating effective DRP5A antibodies?

Several approaches can be used to generate DRP5A-specific antibodies for research:

• Recombinant protein immunization: Expression of full-length or partial DRP5A protein in systems like E. coli, yeast, or baculovirus for immunization . This approach provides a well-defined immunogen but may face challenges with large proteins like DRP5A (~90 kDa).

• Synthetic peptide approach: Selection of unique peptide sequences (typically 15-20 amino acids) from DRP5A regions with minimal similarity to DRP5B, conjugated to carrier proteins for immunization. This method enables targeting of specific domains but may not capture conformational epitopes.

• Phage display technology: Creation of antibody libraries displayed on phage surfaces followed by selection (biopanning) against purified DRP5A . This technique can yield high-affinity antibodies without animal immunization.

For maximum specificity, antibodies should target regions with the least homology to DRP5B, such as portions of the middle domain or the C-terminal region, to minimize cross-reactivity with this close homolog.

What controls are essential when validating a DRP5A antibody?

Comprehensive validation of DRP5A antibodies requires several critical controls:

Validation should include multiple experimental approaches (Western blot, immunofluorescence, immunoprecipitation) to ensure the antibody performs consistently across applications . When conducting studies in drp5a mutants, it's crucial to include wild-type controls processed under identical conditions to accurately assess phenotypic differences .

How can immunofluorescence be optimized to study DRP5A localization during cell division?

Optimized immunofluorescence protocol for DRP5A visualization:

• Sample preparation:

  • Use actively dividing tissues (root tips, shoot apical meristems)

  • Fix in 4% paraformaldehyde in a suitable buffer (e.g., MTSB or PBS)

  • Digest cell walls with cellulase/pectinase for antibody penetration

• Immunolabeling strategy:

  • Permeabilize with 0.1-0.5% Triton X-100

  • Block with 3-5% BSA or normal serum

  • Incubate with validated anti-DRP5A antibody at optimized dilution

  • Use appropriate fluorophore-conjugated secondary antibody

• Co-labeling recommendations:

  • Label with anti-tubulin antibodies to visualize the phragmoplast

  • Include DNA staining (DAPI) to determine mitotic stages

  • Consider co-labeling with cell plate markers

• Imaging considerations:

  • Use confocal microscopy for better resolution

  • Acquire z-stacks to capture complete cell volumes

  • Consider colchicine treatment to enrich for M-phase cells

When analyzing DRP5A localization, it's essential to precisely identify cell cycle stages through appropriate markers, as DRP5A localization changes dramatically from prophase (speckles around the nucleus) to telophase (concentrated at the cell plate) .

What Western blot conditions are optimal for detecting DRP5A in plant tissues?

Optimized Western blot protocol for DRP5A detection:

• Sample preparation:

  • Extract from tissues with high DRP5A expression (meristematic regions)

  • Use extraction buffer containing protease inhibitors

  • Include phosphatase inhibitors to preserve modifications

  • Clear lysates by centrifugation (13,000-15,000 × g)

• Protein separation:

  • Use 8-10% SDS-PAGE gels for optimal separation of DRP5A (~90 kDa)

  • Load 20-50 μg total protein per lane

  • Include appropriate molecular weight markers

• Transfer conditions:

  • Wet transfer: 30V overnight at 4°C

  • Use 0.45 μm PVDF membranes

  • Add 0.1% SDS to transfer buffer to improve large protein transfer

• Antibody incubation:

  • Block with 5% non-fat dry milk or 3% BSA in TBST

  • Incubate with primary antibody overnight at 4°C (1:1000-1:5000)

  • Wash extensively (5 × 5 minutes)

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000)

• Detection:

  • Use enhanced chemiluminescence for sensitive detection

  • Consider fluorescent secondary antibodies for quantification

For troubleshooting weak signals, consider enriching for dividing cells or using colchicine treatment to increase DRP5A abundance, as its expression is limited to M-phase cells .

How can protein-protein interactions involving DRP5A be studied effectively?

Multiple complementary approaches can be used to study DRP5A interactions:

Research has shown that dynamin-related proteins can form both homo-oligomers and hetero-oligomers. For instance, Y2H assays have demonstrated that some plant DRPs can interact with structurally distinct DRPs to form hetero-polymers . When using antibodies for interaction studies, consider whether the antibody epitope might interfere with protein-protein interaction interfaces.

How can contradictory results between antibody localization and GFP-fusion studies be resolved?

When facing discrepancies between antibody-based and GFP-fusion localization patterns for DRP5A, a systematic approach is needed:

• Potential causes of discrepancies:

  • Fixation artifacts in immunostaining

  • GFP tag interference with protein function

  • Expression level differences between native and transgenic proteins

  • Epitope masking in specific protein complexes

  • Temporal dynamics captured differently by each method

• Resolution strategies:

  • Compare multiple fixation methods

  • Test both N- and C-terminal GFP fusions

  • Generate transgenic lines with native promoter expression

  • Perform complementation tests in drp5a mutants with GFP-DRP5A

  • Use multiple antibodies recognizing different epitopes

  • Employ super-resolution microscopy for detailed localization

In studies of DRP5A, both approaches have provided valuable insights. GFP-DRP5A expressed under the DRP5A promoter successfully complements drp5a mutant phenotypes, suggesting functional protein folding and localization . Comparison of DRP5A-GFP fusion protein results with antibody immunofluorescence has shown consistent localization patterns, with both methods revealing speckles and bar-shaped structures during cell division .

How can DRP5A antibodies be used to investigate cell cycle regulation mechanisms?

DRP5A antibodies provide powerful tools for studying cell cycle regulation:

• Expression dynamics analysis:

  • Quantify DRP5A levels across cell cycle stages using immunoblotting

  • Correlate with cell cycle markers to establish precise timing

  • Compare wild-type with cell cycle mutants to identify regulatory pathways

• Cell cycle synchronization studies:

  • Use aphidicolin or colchicine treatments with immunoblotting

  • Track DRP5A abundance after cell cycle release

  • Identify post-translational modifications by mobility shifts

• Post-translational modification mapping:

  • Develop phospho-specific antibodies for DRP5A

  • Combine with phosphatase treatments to confirm specificity

  • Map cell cycle-dependent modifications

• Regulatory pathway analysis:

  • Immunoprecipitate DRP5A to identify cell cycle-dependent interaction partners

  • Examine effects of CDK inhibitors on DRP5A localization

  • Combine with genetic approaches using cell cycle mutants

Research has shown that DRP5A expression is highly regulated during the cell cycle, with M phase-specific accumulation of both mRNA and protein. Colchicine treatment to arrest cells in M phase results in most root tip cells displaying DRP5A fluorescence, while aphidicolin treatment (S-phase arrest) results in minimal signal , confirming tight cell cycle regulation.

How do temperature and environmental stresses affect DRP5A function and localization?

Environmental factors significantly impact DRP5A dynamics:

Understanding how DRP5A responds to environmental stresses provides insights into how plants adapt their cell division machinery to changing conditions. The temperature sensitivity of drp5a mutants suggests that DRP5A function may be particularly important under suboptimal growth conditions .

What is the evolutionary conservation of DRP5A across plant species and how can antibodies help study this?

DRP5A conservation across plant lineages can be investigated using antibodies:

• Cross-species antibody applications:

  • Test reactivity against DRP5A orthologs in diverse plant species

  • Develop antibodies targeting highly conserved epitopes

  • Compare localization patterns during cytokinesis across lineages

• Experimental design for evolutionary studies:

  • Perform side-by-side immunolocalization in related species

  • Combine with cytoskeletal markers to normalize cell division stages

  • Include phylogenetically diverse sampling across major plant groups

• Epitope conservation analysis:

  • Map antibody recognition to sequence conservation patterns

  • Correlate functional constraints with epitope conservation

  • Identify species-specific variations in DRP5A sequences

DRP5A is found in Viridiplantae (green plants), Amoebozoa, and Heterolobosea, suggesting its ancestral role in cytokinesis evolved before the divergence of these lineages . While DRP5A function in cytokinesis appears conserved, specific mechanisms and interactions may vary across plant families, making comparative studies valuable for understanding cytokinesis evolution.

What are common issues in DRP5A antibody experiments and how can they be resolved?

When troubleshooting DRP5A experiments, remember that its expression is highly cell cycle-dependent and restricted to dividing cells, which can lead to challenges in detection in tissues with low division rates .

How can epitope mapping improve DRP5A antibody specificity and application range?

Epitope mapping provides critical information for antibody optimization:

• Mapping techniques:

  • Peptide array analysis using overlapping peptides

  • Deletion/truncation mapping with recombinant fragments

  • Site-directed mutagenesis of predicted epitope regions

  • Hydrogen-deuterium exchange mass spectrometry

• Applications of epitope knowledge:

  • Design more specific antibodies targeting unique regions

  • Develop antibodies recognizing specific conformational states

  • Create phospho-specific antibodies for regulatory studies

  • Engineer cross-species reactive antibodies for evolutionary studies

• Specificity optimization:

  • Create antibodies targeting regions with minimal homology to DRP5B

  • Design epitopes avoiding conserved GTPase domains

  • Develop antibodies recognizing unique post-translational modifications

For DRP5A, targeting regions outside the highly conserved GTPase domain is essential for generating specific antibodies that don't cross-react with DRP5B or other dynamin-related proteins. The middle domain and C-terminal regions typically have lower sequence conservation among dynamin family members and may provide better targets for specific antibody development .

How can DRP5A be accurately quantified across different tissues and developmental stages?

Accurate quantification of DRP5A requires tailored approaches:

• Western blot-based quantification:

  • Use standard curves with recombinant DRP5A protein

  • Employ fluorescent secondary antibodies for linear detection

  • Normalize to stable reference proteins

  • Utilize digital imaging with quantification software

• ELISA development:

  • Create sandwich ELISA using antibodies to different epitopes

  • Develop standard curves with recombinant protein

  • Optimize sample preparation to minimize matrix effects

• Mass spectrometry approaches:

  • Develop selected reaction monitoring (SRM) assays

  • Use isotope-labeled peptide standards for absolute quantification

  • Select DRP5A-specific peptides with good ionization properties

• Flow cytometry for single-cell analysis:

  • Optimize protoplast preparation protocols

  • Perform intracellular staining with DRP5A antibodies

  • Include cell cycle markers for population segregation

When quantifying DRP5A, it's essential to account for its cell cycle-regulated expression pattern. Normalizing to the proportion of dividing cells in each sample is critical for meaningful comparisons between tissues with different rates of cell division .

What methods can assess the functional impact of DRP5A antibodies in live cells?

Several approaches can evaluate how antibodies affect DRP5A function:

• Microinjection studies:

  • Inject DRP5A antibodies into living cells

  • Monitor effects on cytokinesis and cell plate formation

  • Use fluorescently-labeled antibodies to track localization

• Cell-penetrating antibody fragments:

  • Engineer Fab or scFv fragments with cell-penetrating peptides

  • Apply to dividing tissues and observe cytokinesis effects

  • Combine with live cell imaging of cell plate markers

• Experimental design considerations:

  • Include non-specific antibody controls

  • Titrate antibody concentrations to determine dose-response

  • Use multiple antibodies targeting different epitopes

  • Compare effects to genetic knockdowns for validation

• Readout systems:

  • Quantify completed vs. aborted cytokinesis events

  • Measure cell plate formation dynamics

  • Track chromosomal segregation errors

  • Assess multinucleation as indicator of cytokinesis failure

When designing such experiments, it's important to consider whether the antibody epitope corresponds to functionally important domains of DRP5A, such as the GTPase domain or regions involved in protein-protein interactions. Antibodies targeting different functional domains may produce distinct phenotypic effects .