DRP1C Antibody

What is DRP1C and what cellular processes does it participate in?

DRP1C is a member of the Arabidopsis dynamin-related protein 1 (DRP1) subfamily that plays essential roles in polarized cell expansion, cytokinesis, and plasma membrane maintenance. DRP1C localizes to the division plane in dividing cells and to the plasma membrane in expanding interphase cells . It forms dynamic foci at the cell cortex in both tip-growing root hairs and diffuse-polar expanding epidermal cells .

DRP1C is particularly important for:

• Pollen development (drp1C-1 mutants exhibit male gametophytic lethality with shriveled, non-germinating pollen)

• Plasma membrane maintenance (mutant pollen shows large furrows and undulations of plasma membrane)

• Clathrin-mediated endocytosis (CME) based on its colocalization with clathrin light chain)

How does DRP1C differ from other members of the DRP family in plants?

The plant DRP family contains structurally distinct subfamilies with different functions:

• DRP1 subfamily (including DRP1C): Plant-specific DRPs that lack some domains found in animal dynamins

• DRP2 subfamily: Contains proteins structurally similar to animal dynamins

Key differences:

• DRP1C interacts with DRP1A but these proteins exhibit different responses to endocytic inhibitors

• DRP1C cannot functionally complement DRP1A mutations, suggesting unique roles

• While some DRPs (like DRP3A) can self-interact, DRP2B doesn't interact with itself but does interact with DRP1A

• The five DRP1 proteins (DRP1A-E) have distinct in planta roles due to differences in their spatiotemporal expression patterns

What methods are used to visualize DRP1C localization in plant cells?

Several microscopy techniques have been employed to study DRP1C localization:

How can I establish the functionality of a DRP1C-GFP fusion protein?

To verify that a DRP1C-GFP fusion is functional and suitable for localization studies:

• Genetic complementation: Transform drp1C-1 mutants with the DRP1C-GFP construct and check for rescue of the male gametophytic lethal phenotype

• Expression level assessment: Perform immunoblot analysis of total protein extracts to confirm that the fusion protein is intact and expressed at levels similar to native DRP1C

• Subcellular localization: Verify that the fusion protein localizes to expected structures (division plane in dividing cells, plasma membrane in expanding cells)

• Dynamic behavior: Confirm that DRP1C-GFP forms the characteristic dynamic foci at the cell cortex that colocalize with clathrin light chain

How do the dynamics of DRP1C foci at the plasma membrane compare to those of other DRPs?

The dynamics of DRP1 proteins at the plasma membrane have been characterized through extensive live-cell imaging studies:

These differences suggest that despite their involvement in similar cellular processes, DRP1C and DRP1A have distinct molecular properties .

What experimental approaches can determine the recruitment mechanism of DRP1C to the plasma membrane?

Fluorescence Recovery After Photobleaching (FRAP) analysis has revealed important insights about DRP1C recruitment:

• Cytoplasmic pool recovery: The cytoplasmic pool of DRP1C-GFP recovered to original intensity in 17.1 ± 5.1 s

• Plasma membrane-associated recovery: Plasma membrane-associated fluorescence recovered in 47.6 ± 15.8 s, indicating that DRP1C-GFP does not freely diffuse at the plasma membrane

• Regional recovery analysis: By dividing the photobleached area into peripheral and inner regions, researchers determined that in 83% of root hairs, both regions recovered with equal kinetics, suggesting DRP1C-GFP is primarily recruited directly from the cytoplasm rather than through lateral diffusion along the membrane

Additional approaches could include:

• Pharmacological inhibition of different trafficking pathways

• Genetic manipulation of potential recruitment factors

• Single-particle tracking to analyze individual DRP1C molecule behavior

How do inhibitors of cellular processes affect DRP1C localization and function?

Various inhibitors have been used to investigate the requirements for DRP1C localization and dynamics:

These results suggest that DRP1C localization at the tip of root hairs is intimately tied with active growth, but the requirements for active root hair tip growth are likely not directly required for DRP1C-GFP recruitment or dynamics at the plasma membrane .

What mechanisms underlie the interaction between DRP1C and the clathrin endocytic machinery?

The relationship between DRP1C and clathrin-mediated endocytosis (CME) involves complex molecular interactions:

• Colocalization: DRP1C-GFP forms dynamic foci at the cell cortex that colocalize with clathrin light chain fluorescent fusion protein (CLC-FFP), suggesting participation in clathrin-mediated membrane dynamics

• Temporal dynamics: Time-lapse VAEM observations showed that DRP1C appears and accumulates on existing CLC foci and disappears at the same time as or immediately after the disappearance of CLC

• Inhibitor sensitivity: The CME inhibitor tyrphostin A23 dramatically affects DRP1C dynamics, supporting its involvement in the clathrin pathway

• Protein interactions: Beyond clathrin, DRP1C likely interacts with other proteins involved in endocytosis. For instance, DRP1C interacts with DRP1A, which also participates in CME

• Structural role: By analogy with animal dynamins, DRP1C might participate in the constriction and scission of clathrin-coated vesicles from the plasma membrane

How should experiments be designed to distinguish between redundant and unique functions of DRP1C compared to other DRPs?

To differentiate between redundant and unique functions of DRP1C:

• Genetic approaches:

  • Analyze phenotypes of single drp1c mutants vs. double/multiple drp mutants

  • Create chimeric proteins by domain swapping between DRP1C and other DRPs

  • Perform cross-complementation studies (expression of one DRP under another DRP's promoter)

• Biochemical characterization:

  • Compare GTPase activities and membrane binding properties of purified proteins

  • Investigate protein-protein interactions through yeast two-hybrid assays or co-immunoprecipitation

  • Analyze post-translational modifications specific to each DRP

• Advanced imaging:

  • Use dual-color imaging to track dynamics of multiple DRPs simultaneously

  • Apply super-resolution microscopy to resolve subtle differences in localization

  • Perform correlative light and electron microscopy to connect dynamic behavior with ultrastructural context

• Tissue/cell type-specific analysis:

  • Compare DRP1C function in different cell types (e.g., pollen vs. root hairs)

  • Use tissue-specific promoters to express DRP1C in specific cell types of multiple drp mutants

What controls should be included when using DRP1C antibodies for immunolocalization studies?

When performing immunolocalization with DRP1C antibodies:

• Negative controls:

  • drp1c knockout mutant tissues (should show no signal)

  • Primary antibody omission

  • Pre-immune serum in place of primary antibody

  • Peptide competition assay (pre-incubation of antibody with immunizing peptide)

• Positive controls:

  • Tissues known to express high levels of DRP1C (e.g., pollen, root hairs)

  • Samples from plants overexpressing DRP1C

  • Parallel detection of DRP1C-GFP using anti-GFP antibodies in transgenic plants

• Validation of specificity:

  • Western blot analysis to confirm antibody recognizes a single band of appropriate molecular weight

  • Comparative immunolabeling with antibodies raised against different epitopes of DRP1C

  • Cross-reactivity assessment with other DRP family members

How can I optimize live-cell imaging protocols for studying DRP1C dynamics?

For optimal live-cell imaging of DRP1C:

• Construct design considerations:

  • Use the native DRP1C promoter (2.9 kb upstream sequence has been shown to complement drp1C mutation)

  • Position the fluorescent tag to minimize functional interference

  • Consider photobleaching characteristics (DRP1C-GFP shows less photobleaching compared to DRP1A-GFP)

• Imaging parameters:

  • For plasma membrane-associated events, VAEM provides superior resolution compared to conventional CLSM

  • Minimize laser power and exposure time to reduce photobleaching and phototoxicity

  • Optimize frame rate (typically 1-2 frames/second) to capture the dynamics of DRP1C foci

• Sample preparation:

  • Image seedlings directly growing on microscope slides with appropriate growth medium

  • Maintain stable temperature and humidity during imaging

  • For root hair imaging, ensure plants are actively growing with minimal mechanical stress

• Analysis approaches:

  • Track individual foci to determine lifetime, movement patterns, and intensity changes

  • Measure colocalization with other proteins quantitatively (e.g., Pearson's correlation coefficient)

  • Analyze photobleaching kinetics to infer molecular dynamics and exchange rates

What are the key considerations for analyzing mutant phenotypes to understand DRP1C function?

When analyzing drp1c mutant phenotypes:

• Genetic background issues:

  • Use multiple independent alleles or complementation tests to confirm phenotype specificity

  • Consider using CRISPR/Cas9 to generate new alleles in different backgrounds

  • For male gametophytic lethal phenotypes, heterozygotes should show ~50% aborted pollen

• Cell-specific analyses:

  • For pollen development, examine membrane organization using transmission electron microscopy

  • For vegetative tissues, focus on cell types where DRP1C is highly expressed

  • Analyze dynamics of other cellular processes (e.g., endocytosis, secretion) using appropriate markers

• Temporal considerations:

  • Examine phenotypes at different developmental stages

  • Use inducible systems to distinguish between developmental and physiological roles

• Functional redundancy:

  • Generate and analyze double/multiple mutants with other DRPs

  • Use tissue-specific or inducible knockdown approaches for genes with lethal null phenotypes

  • Consider dosage effects in heterozygotes or weak alleles

How can I address difficulties in observing DRP1C dynamics in specific cell types?

When facing challenges in visualizing DRP1C:

• Expression level optimization:

  • If native promoter gives weak signals, consider using stronger promoters while ensuring complementation

  • For transient expression, optimize promoter-terminator combinations

  • Test different fluorophores with optimal brightness and photostability for plant cells

• Cell type-specific considerations:

  • For tip-growing cells (pollen tubes, root hairs), ensure active growth during imaging

  • For thick tissues, use proper mounting techniques or tissue clearing methods

  • Use tissue-specific promoters to enhance expression in cells of interest

• Technical approaches:

  • Optimize microscope settings for signal-to-noise ratio (pinhole size, detector gain, laser power)

  • Consider using spinning disk confocal or light sheet microscopy for reduced photobleaching

  • Apply deconvolution algorithms to improve image quality

• Alternative visualization strategies:

  • Use split-fluorescent protein systems for in vivo interaction studies

  • Consider FRET-based approaches to study DRP1C interactions with membrane or proteins

  • Use photoconvertible fluorescent proteins to track subpopulations of DRP1C molecules

What strategies can resolve contradictory results between in vitro biochemical assays and in vivo observations of DRP1C?

When encountering discrepancies between in vitro and in vivo results:

• Protein preparation considerations:

  • Ensure purified proteins maintain native conformation and activity

  • Compare bacterially-expressed proteins with those purified from plant systems

  • Consider the impact of tags used for protein purification

• Experimental conditions:

  • Adjust in vitro conditions to better mimic cellular environment (pH, ion concentrations, membrane composition)

  • Include relevant cofactors and interacting proteins in biochemical assays

  • Test activity under varying nucleotide concentrations

• Bridging approaches:

  • Use semi-in vitro systems (e.g., isolated plasma membrane vesicles with purified proteins)

  • Perform structure-function analyses with site-directed mutagenesis both in vitro and in vivo

  • Develop quantitative assays that can be applied both in vitro and in vivo

• Differential regulation:

  • Investigate post-translational modifications that might occur in vivo but not in vitro

  • Consider protein complex formation that might alter activity

  • Examine the influence of membrane composition and curvature on activity

What emerging technologies might advance our understanding of DRP1C function?

Several cutting-edge approaches could propel DRP1C research forward:

• Advanced imaging technologies:

  • Super-resolution microscopy (PALM/STORM, SIM, STED) to resolve DRP1C organization beyond the diffraction limit

  • Single-molecule tracking to analyze DRP1C behavior at the individual molecule level

  • Correlative light and electron microscopy to connect dynamic behavior with ultrastructural context

• Protein engineering approaches:

  • Optogenetic tools to spatiotemporally control DRP1C activity

  • Biosensors to monitor DRP1C conformation changes or GTPase activity in vivo

  • Engineered DRP1C variants with altered properties to dissect structure-function relationships

• Omics integration:

  • Proteomics to identify the complete DRP1C interactome under different conditions

  • Phosphoproteomics to characterize regulatory post-translational modifications

  • Systems biology approaches to place DRP1C in broader cellular networks

• In situ structural biology:

  • Cryo-electron tomography of cellular structures containing DRP1C

  • In-cell NMR to study DRP1C dynamics in native environment

  • Proximity labeling (BioID, APEX) to map molecular neighborhoods of DRP1C

How might understanding DRP1C function contribute to applications in plant biotechnology?

Knowledge of DRP1C biology could lead to numerous applications:

• Crop improvement strategies:

  • Engineering membrane trafficking dynamics to enhance stress tolerance

  • Modifying pollen development and fertility for hybrid seed production

  • Optimizing cell expansion for biomass production or specific tissue properties

• Biotechnological tools:

  • Developing DRP1C-based tools for controlled vesicle formation in plant cell factories

  • Creating biosensors for monitoring membrane dynamics in response to environmental stimuli

  • Engineering chimeric proteins with novel membrane remodeling properties

• Fundamental insights:

  • Understanding plant-specific adaptations in membrane trafficking

  • Elucidating evolutionary relationships between plant and animal endocytic machineries

  • Revealing mechanisms of cell polarity establishment and maintenance in plants

• Environmental applications:

  • Developing plants with enhanced uptake capabilities for phytoremediation

  • Engineering root systems with optimized nutrient acquisition properties

  • Creating crops with improved drought resistance through membrane trafficking modulation