EXO70E2 Antibody

What is EXO70E2 and why is it important for plant cell biology?

EXO70E2 is a member of the EXO70 gene family, which encodes putative exocyst subunits that are conserved in land plants. Specifically, EXO70E2 functions as an essential component of the exocyst complex, playing a critical role in mediating unconventional protein secretion in plants through exocyst-positive organelles (EXPOs) . Arabidopsis thaliana contains 23 putative EXO70 genes that can be classified into eight phylogenetic clusters . Among these paralogs, EXO70E2 has been identified as uniquely important because it serves as a nucleation point for recruiting other exocyst subunits to EXPO formation . Unlike some other EXO70 paralogs that function in conventional exocytosis or autophagy pathways, EXO70E2 specifically participates in unconventional protein secretion mechanisms, making it a key protein for understanding alternative secretory pathways in plants .

How does EXO70E2 differ from other EXO70 family proteins?

EXO70E2 possesses unique functional characteristics that distinguish it from other EXO70 family members:

• Recruitment capacity: EXO70E2 has the specific ability to recruit several exocyst subunits (especially Sec family members) to EXPO formation sites, a capability not shared by other paralogs like EXO70A1 .

• Subcellular localization: When expressed in plant cells, EXO70E2 localizes to discrete punctate structures at the plasma membrane and in the cytoplasm, representing EXPOs, while other paralogs may show diffuse cytosolic distribution or localize to different compartments .

• Cross-species functionality: Uniquely, AtEXO70E2 can induce EXPO-like structure formation even when expressed in animal cells (HEK293A), whereas yeast and human Exo70 homologs cannot induce EXPO formation in Arabidopsis protoplasts .

• Interaction network: EXO70E2 specifically interacts with select exocyst components as demonstrated by FRET and BiFC analyses, showing direct interaction with Sec6 and Sec10 but not with Sec8, Sec3a, Sec5a, or Sec15b .

This functional specialization makes EXO70E2 a distinct member within the large EXO70 family with a specific role in unconventional secretion pathways.

What are the optimal conditions for immunolocalization of EXO70E2 in plant tissues?

For effective immunolocalization of EXO70E2 in plant tissues, researchers should follow these methodological guidelines:

• Fixation: Use 4% paraformaldehyde in phosphate buffer (pH 7.2) for 2-4 hours at room temperature for tissue preservation while maintaining antigen accessibility.

• Sectioning: For confocal microscopy, 50-100 μm vibratome sections are recommended; for higher resolution studies, prepare 70-90 nm ultrathin sections for immunogold electron microscopy.

• Blocking: Use 3% BSA in PBS with 0.1% Triton X-100 for 1-2 hours to reduce non-specific binding.

• Primary antibody incubation: Apply anti-EXO70E2 antibody at 1:100-1:500 dilution (optimization required) and incubate overnight at 4°C .

• Controls: Always include negative controls (omitting primary antibody) and positive controls (tissues known to express EXO70E2).

• Detection system: For immunofluorescence, use fluorophore-conjugated secondary antibodies; for electron microscopy, gold-conjugated secondary antibodies (typically 10-15 nm gold particles) have been successfully employed to visualize EXO70E2 at EXPO membranes and fusion sites .

When studying EXO70E2 localization at the ultrastructural level, immunogold labeling has revealed concentrated signals at plasma membrane fusion sites where EXPO outer membranes connect with the PM during secretion events .

How can I validate the specificity of an EXO70E2 antibody?

Validating EXO70E2 antibody specificity is crucial for reliable experimental results. A comprehensive validation approach includes:

• Western blot analysis: Run protein extracts from wild-type tissues alongside exo70e2 mutant samples or tissues overexpressing EXO70E2. A specific antibody should detect a single band at the predicted molecular weight (~70 kDa) in wild-type and overexpression samples, with reduced or absent signal in mutants .

• Immunoprecipitation: Perform immunoprecipitation followed by mass spectrometry to confirm that the antibody pulls down EXO70E2 and its known interacting partners like Sec6 and Sec10 .

• Heterologous expression systems: Express tagged versions of EXO70E2 (e.g., GFP-tagged) in systems like HEK293A cells and confirm antibody recognition by both Western blot and immunofluorescence, comparing signals from anti-GFP and anti-EXO70E2 antibodies .

• Cross-reactivity testing: Test reactivity against multiple EXO70 paralogs to ensure the antibody doesn't cross-react with closely related family members, particularly EXO70E1 which shares sequence similarity.

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide before immunostaining or Western blotting - a specific antibody's signal should be significantly reduced or eliminated.

The search results indicate that EXO70E2 antibodies have been successfully used in immunogold electron microscopy to specifically label EXPO structures in plant cells and EXPO-like structures in HEK cells expressing AtEXO70E2-GFP .

How can EXO70E2 antibodies be used to study the dynamics of EXPO formation?

EXO70E2 antibodies provide powerful tools for investigating EXPO formation dynamics through these methodological approaches:

• Live-cell immunolabeling: Using membrane-permeable fluorescently-labeled EXO70E2 antibody fragments to track EXPO dynamics in living cells, complementing GFP-fusion protein studies.

• Pulse-chase immunolabeling: Apply timed immunolabeling at different stages of cell development or following stimuli to capture the temporal sequence of EXPO formation.

• Co-immunoprecipitation with time-course analysis: Use EXO70E2 antibodies for immunoprecipitation at different time points following cellular stimulation to identify the sequential recruitment of exocyst components, based on the finding that EXO70E2 serves as a recruitment nucleus for other exocyst subunits .

• Super-resolution microscopy: Combine EXO70E2 antibody labeling with techniques like STORM or PALM to visualize EXPO formation at nanoscale resolution, revealing structural details beyond conventional light microscopy.

• Correlative light-electron microscopy (CLEM): Use EXO70E2 antibodies to first identify EXPOs by fluorescence microscopy, then examine the same structures by electron microscopy to correlate functional dynamics with ultrastructural changes.

Studies have demonstrated that EXO70E2 is essential for recruiting exocyst subunits like Sec5a, Sec6, Sec8, and Sec10 to EXPO formation sites. When expressed alone, these subunits show diffuse cytosolic signals, but they localize to discrete punctate structures when co-expressed with EXO70E2 .

What methodological approaches can be used to study the molecular interactions between EXO70E2 and other exocyst components?

To investigate molecular interactions between EXO70E2 and other exocyst components, researchers can employ these sophisticated methodological approaches:

• FRET (Fluorescence Resonance Energy Transfer): This technique has successfully demonstrated direct interactions between EXO70E2 and specific exocyst components by measuring energy transfer between fluorophore-tagged proteins. Spectral FRET analysis revealed significant shifts from CFP to YFP emission peaks when examining EXO70E2 interactions with other exocyst subunits .

• BiFC (Bimolecular Fluorescence Complementation): By tagging potential interaction partners with split fluorescent protein fragments (YC and YN), researchers have visualized direct protein-protein interactions through complementation when EXO70E2 interacts with certain exocyst subunits .

• Yeast two-hybrid assays: This can be used as a complementary approach to screen for potential interacting partners of EXO70E2.

• In vitro binding assays: Using purified recombinant proteins to assess direct binding between EXO70E2 and other exocyst components under controlled conditions.

• Cross-linking mass spectrometry: This approach can identify interaction interfaces between EXO70E2 and its binding partners at the amino acid resolution level.

The interaction network based on FRET and BiFC analyses (summarized in Table 1) shows the following pattern of direct interactions:

This interaction map demonstrates that EXO70E2 directly interacts with Sec6 and Sec10, which then serve as bridging molecules to recruit other exocyst components .

How can I design experiments to distinguish between EXO70E2-mediated conventional and unconventional secretion pathways?

Designing experiments to differentiate between EXO70E2-mediated conventional and unconventional secretion pathways requires sophisticated approaches:

• Cargo protein selection:

  • For conventional pathway: Use proteins with signal peptides that traffic through ER-Golgi (e.g., secGFP)

  • For unconventional pathway: Use leaderless secretory proteins known to be secreted via EXPO (e.g., SAMS2)

• Pharmacological treatments:

  • Apply Brefeldin A to disrupt ER-Golgi transport and observe which secretion routes are affected

  • Use cytoskeleton disruptors (latrunculin B, oryzalin) to determine cytoskeletal requirements for each pathway

• Temperature blocks:

  • Employ temperature shifts (e.g., 15°C blocks) that selectively impact conventional secretion

  • Monitor secretion of marker proteins under these conditions

• Genetic approaches:

  • Generate EXO70E2 knockouts/knockdowns and assess effects on both pathways

  • Create chimeric proteins fusing EXO70E2 domains with other EXO70 family members to identify regions responsible for unconventional secretion specificity

• Advanced imaging:

  • Perform dual-color live cell imaging using differently labeled cargo proteins for each pathway

  • Use EXPO-specific markers alongside conventional secretory pathway markers

Research has shown that EXPOs are distinct from autophagosomes, as they do not label with the standard autophagosomal marker Atg8E, and appear to be specifically involved in unconventional protein secretion rather than degradative pathways .

What are common problems in EXO70E2 antibody-based experiments and how can they be resolved?

Researchers frequently encounter these challenges when working with EXO70E2 antibodies, along with recommended solutions:

• High background signal:

  • Problem: Non-specific binding producing excessive background in immunostaining.

  • Solution: Optimize blocking conditions using 5% BSA or normal serum matching the secondary antibody host; include 0.1-0.3% Triton X-100 for permeabilization; extend blocking time to 2 hours at room temperature; perform additional washing steps with 0.1% Tween-20 in PBS .

• Weak or absent signal:

  • Problem: Insufficient detection of EXO70E2.

  • Solution: Verify antibody reactivity against recombinant EXO70E2; optimize antibody concentration (try 1:50 to 1:500 dilutions); improve antigen retrieval methods for fixed tissues; use signal amplification systems like tyramide signal amplification; store antibody properly to maintain activity .

• Cross-reactivity with other EXO70 paralogs:

  • Problem: Antibody recognizes multiple EXO70 family members.

  • Solution: Pre-adsorb antibody with recombinant proteins of closely related paralogs; validate in exo70e2 mutant tissues; use monoclonal antibodies targeting unique epitopes; perform Western blots to confirm specificity.

• Inconsistent results between experiments:

  • Problem: Variable staining patterns or detection efficiency.

  • Solution: Standardize fixation protocols; use consistent antibody lots; prepare fresh working dilutions for each experiment; include internal positive controls; standardize image acquisition settings.

• Poor penetration in thick tissue samples:

  • Problem: Inadequate staining in tissue depth.

  • Solution: Optimize permeabilization; extend antibody incubation times; use thinner sections; consider vibratome sectioning followed by free-floating antibody incubation; use tissue clearing methods compatible with immunostaining.

Remember that EXO70E2 antibodies have been successfully employed in immunogold electron microscopy studies, which require specific sample preparation and labeling protocols that differ from standard immunofluorescence approaches .

How should EXO70E2 antibody be stored and handled to maintain optimal activity?

Proper storage and handling of EXO70E2 antibodies are critical for maintaining their performance over time:

• Storage conditions:

  • Store lyophilized antibody at -20°C upon receipt until ready for reconstitution

  • After reconstitution, store working aliquots at -20°C (for frequent use) or -80°C (for long-term storage)

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Reconstitution guidelines:

  • Reconstitute lyophilized antibody in sterile water or recommended buffer

  • Allow complete dissolution by gentle mixing rather than vortexing

  • Let stand at room temperature for 5-10 minutes before aliquoting

• Working dilution preparation:

  • Prepare fresh working dilutions on the day of experiment

  • Dilute in buffer containing 1-3% BSA or appropriate carrier protein

  • Include 0.01-0.05% sodium azide in stored antibody solutions to prevent microbial growth

• Transportation precautions:

  • Transport on ice or with cold packs

  • The product is shipped at 4°C but should be stored at recommended temperatures immediately upon receipt

• Quality control measures:

  • Periodically test antibody activity against positive control samples

  • Monitor for signs of degradation (precipitation, cloudy appearance)

  • Record lot numbers and correlate with experimental outcomes

• Contamination prevention:

  • Use sterile technique when handling antibody solutions

  • Avoid introducing bacteria or fungi by using sterile pipette tips and containers

  • Filter buffers used for dilution through 0.22 μm filters

Following these guidelines will help ensure consistent results across experiments and maximize the useful lifetime of EXO70E2 antibodies.

How can EXO70E2 antibodies be used to investigate the role of EXPO in plant stress responses?

EXO70E2 antibodies provide valuable tools for investigating EXPO's role in plant stress response through several methodological approaches:

• Stress-induced expression profiling:

  • Apply various stressors (drought, salt, pathogen, heat) to plant tissues

  • Use EXO70E2 antibodies for immunoblotting to quantify expression changes

  • Correlate EXO70E2 levels with stress intensity and duration

• Subcellular redistribution analysis:

  • Employ immunofluorescence microscopy to track changes in EXO70E2 localization patterns during stress

  • Quantify EXPO numbers and distribution before and after stress application

  • Co-localize with stress-induced secreted proteins lacking signal peptides

• EXPO cargo identification during stress:

  • Immunoprecipitate EXO70E2-containing structures from stressed tissues

  • Analyze associated proteins by mass spectrometry

  • Compare EXPO cargo profiles between normal and stress conditions

• Genetic interaction studies:

  • Generate plant lines with modified EXO70E2 expression in stress-response mutant backgrounds

  • Use antibodies to verify expression levels

  • Assess phenotypic consequences and stress tolerance

• EXPO fusion events monitoring:

  • Use immunogold electron microscopy to quantify EXPO-plasma membrane fusion events during stress

  • Correlate fusion frequency with stress intensity

Research indicates that EXPOs mediate unconventional protein secretion, which is often upregulated during stress conditions . The distinct double-membrane structure of EXPOs, visualized by immunogold labeling with EXO70E2 antibodies, suggests a specialized secretory function that may be particularly important during environmental challenges when conventional secretory pathways might be compromised .

What experimental approaches can determine if EXO70E2 function is conserved across different plant species?

To determine if EXO70E2 function is conserved across plant species, researchers can employ these methodological approaches:

• Comparative genomics and phylogenetic analysis:

  • Identify EXO70E2 orthologs across plant lineages

  • Analyze sequence conservation, particularly in functional domains

  • Construct phylogenetic trees to establish evolutionary relationships

• Cross-species antibody reactivity testing:

  • Test EXO70E2 antibodies against protein extracts from diverse plant species

  • Validate specificity through Western blotting and immunoprecipitation

  • Compare band patterns to assess protein size conservation

• Heterologous complementation assays:

  • Express EXO70E2 from different species in Arabidopsis exo70e2 mutants

  • Use antibodies to confirm expression

  • Assess functional rescue of EXPO formation and unconventional secretion

• Cross-species localization studies:

  • Perform immunolocalization with EXO70E2 antibodies in multiple plant species

  • Compare subcellular distribution patterns

  • Identify conserved structures resembling EXPOs

• Protein interaction network comparison:

  • Use co-immunoprecipitation with EXO70E2 antibodies in different species

  • Analyze interaction partners by mass spectrometry

  • Compare exocyst component binding profiles across species

Research has shown that EXO70E2 has a unique ability to induce EXPO formation even in animal cells (HEK293A), suggesting fundamental conservation of its membrane-binding properties beyond the plant kingdom . Electron microscopy confirmed that AtEXO70E2 expression in HEK cells induced double-membraned, EXPO-like structures that were recognized by both GFP and AtEXO70E2 antibodies .

How can advanced imaging techniques be combined with EXO70E2 antibodies to reveal new insights about EXPO biogenesis?

Combining cutting-edge imaging techniques with EXO70E2 antibodies can reveal unprecedented details about EXPO biogenesis:

• Super-resolution microscopy approaches:

  • STORM/PALM: Use photo-switchable fluorophore-conjugated EXO70E2 antibodies to achieve 20-30 nm resolution of EXPO structures

  • STED microscopy: Apply to visualize the precise arrangement of EXO70E2 during initial EXPO formation

  • SIM: Employ structured illumination to improve resolution 2-fold beyond conventional microscopy while maintaining live-cell compatibility

• Correlative Light and Electron Microscopy (CLEM):

  • Identify EXO70E2-positive structures by fluorescence microscopy

  • Process the same sample for electron microscopy

  • Use immunogold labeling with EXO70E2 antibodies

  • Correlate fluorescence patterns with ultrastructural features at nanometer resolution

• Live-cell imaging with antibody fragments:

  • Generate fluorescently-labeled Fab fragments of EXO70E2 antibodies

  • Introduce into living cells via microinjection or cell-penetrating peptide conjugation

  • Track EXPO dynamics in real-time without fixation artifacts

• Expansion microscopy:

  • Apply physical tissue expansion techniques before immunolabeling

  • Achieve improved spatial resolution with standard confocal microscopy

  • Preserve relative protein positions while increasing their separation

• Cryo-electron tomography:

  • Vitrify cells expressing EXO70E2

  • Perform immunogold labeling on cryosections

  • Generate 3D tomograms of EXPO structures at molecular resolution

• Light-sheet microscopy:

  • Visualize EXO70E2-labeled structures across entire tissues with minimal photodamage

  • Observe EXPO distribution and dynamics at the organ level

Electron microscopy studies have already revealed that EXPOs are double-membrane organelles that can fuse with the plasma membrane, showing continuity between the outer EXPO membrane and the PM during fusion events . These structures can be specifically labeled with EXO70E2 antibodies at their membrane, confirming EXO70E2's role in defining these organelles .

How can researchers experimentally differentiate the functions of EXO70E2 from other EXO70 paralogs?

To experimentally differentiate the functions of EXO70E2 from other EXO70 paralogs, researchers can implement these systematic approaches:

• Paralog-specific knockout/knockdown studies:

  • Generate single and combinatorial mutants of different EXO70 paralogs

  • Use EXO70E2 antibodies to confirm specificity of targeting

  • Compare phenotypic consequences and cellular effects

• Domain swap experiments:

  • Create chimeric proteins exchanging domains between EXO70E2 and other paralogs

  • Determine which domains confer specific functions (EXPO formation, protein recruitment)

  • Analyze localization and functional capacity of chimeras using antibodies

• Differential interactome analysis:

  • Perform parallel immunoprecipitations of different EXO70 paralogs

  • Compare interaction partners by mass spectrometry

  • Identify unique and shared binding proteins

• Subcellular localization mapping:

  • Compare localization patterns of multiple EXO70 paralogs simultaneously

  • Perform triple-labeling experiments with EXO70E2 and other family members

  • Quantify colocalization coefficients

Research has already demonstrated functional specialization within the EXO70 family:

• EXO70E2 specifically localizes to EXPO structures and is essential for unconventional protein secretion

• EXO70A1 and EXO70B2 are more involved in conventional exocytic events

• EXO70B1 associates with autophagy pathways leading to tonoplast fusion

When tested for recruitment capacity, EXO70E2 could successfully recruit Sec family members to punctate structures, while some other paralogs (EXO70A3, C1, D1-3, F1, H2, H4, H6, H8) remained cytosolic even when co-expressed with EXO70E2 . This demonstrates the unique protein interaction capabilities of EXO70E2 compared to other family members.

What are the methodological considerations for using antibodies to study multiple EXO70 paralogs simultaneously?

When using antibodies to study multiple EXO70 paralogs simultaneously, researchers must address several methodological challenges:

• Antibody specificity validation:

  • Test each antibody against recombinant proteins of all paralogs to assess cross-reactivity

  • Validate in corresponding knockout/knockdown lines

  • Perform peptide competition assays to confirm epitope specificity

• Multiplexed immunodetection strategies:

  • Select antibodies raised in different host species to enable simultaneous detection

  • Use isotype-specific secondary antibodies when primary antibodies come from the same species

  • Employ sequential labeling with careful blocking between steps when using antibodies from the same host

• Signal discrimination approaches:

  • Select fluorophores with minimal spectral overlap for immunofluorescence

  • Implement spectral unmixing algorithms during image acquisition and processing

  • Consider quantum dots with narrow emission spectra for multiplexed detection

• Controls for co-detection experiments:

  • Include single-antibody controls to assess bleed-through

  • Test antibodies individually before combining

  • Include specimens known to express specific paralogs as positive controls

• Quantitative analysis methods:

  • Establish standardized intensity thresholds for each antibody

  • Use appropriate colocalization metrics (Pearson's, Manders' coefficients)

  • Implement computational approaches to distinguish partially overlapping signals

Research investigating multiple EXO70 paralogs has demonstrated that they can localize to different subcellular compartments and participate in distinct cellular processes. For example, while EXO70E2 specifically marks EXPO structures involved in unconventional secretion, other paralogs like EXO70A1 function in conventional secretion pathways, and EXO70B1 participates in autophagy mechanisms .

What methodological innovations could advance our understanding of EXO70E2 function in plant-microbe interactions?

Several methodological innovations could significantly advance our understanding of EXO70E2's role in plant-microbe interactions:

• Cell-specific EXO70E2 manipulation techniques:

  • Develop tissue-specific or inducible EXO70E2 expression/knockout systems

  • Create cell-type specific promoter-driven EXO70E2 variants

  • Monitor using paralog-specific antibodies to track expression patterns during infection

• Advanced imaging at the plant-microbe interface:

  • Implement 4D live-cell super-resolution microscopy to visualize EXO70E2-labeled structures during pathogen attack

  • Use correlative light-electron microscopy with immunogold labeling to examine EXPO structures at infection sites

  • Develop FRET-based biosensors to detect EXO70E2 activation during microbial interactions

• EXPO cargo identification during infection:

  • Apply proximity labeling methods (BioID, APEX) fused to EXO70E2 to identify proteins in its vicinity during infection

  • Use EXO70E2 antibodies to immunoprecipitate complexes from infected tissues

  • Perform comparative proteomics between healthy and infected tissues

• Single-cell approaches:

  • Implement single-cell transcriptomics with spatial resolution to map EXO70E2 expression patterns around infection sites

  • Combine with antibody-based protein detection methods for correlation between transcript and protein levels

  • Develop microfluidic devices to study EXO70E2-dependent secretion in isolated plant cells during microbial challenge

• Structural biology approaches:

  • Determine the atomic structure of EXO70E2 through X-ray crystallography or cryo-EM

  • Map binding interfaces with microbial effectors

  • Design structure-based inhibitors or enhancers of EXO70E2 function

Research has already established that unconventional protein secretion pathways involving EXPOs may be particularly important during stress conditions, including pathogen attack . The ability of EXO70E2 to form double-membrane organelles capable of fusing with the plasma membrane suggests a specialized secretory mechanism that could deliver antimicrobial compounds or immune signaling molecules during plant-microbe interactions .

How might synthetic biology approaches utilizing EXO70E2 antibodies advance our understanding of unconventional protein secretion?

Synthetic biology approaches incorporating EXO70E2 antibodies could transform our understanding of unconventional protein secretion through these innovative methodologies:

• Engineered antibody-based biosensors:

  • Develop split-fluorescent protein systems where one half is fused to an EXO70E2-targeting antibody fragment

  • Create FRET-based sensors using antibody fragments to detect conformational changes in EXO70E2 during EXPO formation

  • Design synthetic circuits that activate reporter genes when antibodies detect EXO70E2 recruitment

• Optogenetic control of EXO70E2 function:

  • Create light-inducible EXO70E2 inhibitory antibody fragments

  • Develop optogenetic tools to trigger EXO70E2 clustering and EXPO formation

  • Use antibodies to validate system functionality and specificity

• Minimal EXPO reconstitution systems:

  • Identify essential components for EXPO formation using EXO70E2 as a foundation

  • Verify proper assembly using specific antibodies

  • Create synthetic membrane systems with purified components to reconstitute EXPO biogenesis in vitro

• Engineered cargo targeting:

  • Design synthetic proteins with EXPO-targeting signals

  • Create antibody-based detection systems to monitor unconventional secretion efficiency

  • Develop therapeutic protein delivery systems exploiting the EXPO pathway

• Orthogonal EXO70 systems:

  • Engineer modified EXO70E2 variants that interact with specific partners

  • Create parallel secretion pathways for different cargo classes

  • Use paralog-specific antibodies to track and validate the orthogonal systems

Research has demonstrated that EXO70E2 has the remarkable ability to induce EXPO-like structures even when expressed in animal cells (HEK293A), suggesting fundamental conservation of its membrane-binding properties . This cross-kingdom functionality presents exciting opportunities for developing synthetic biology tools that could function in diverse cellular contexts.

What are the emerging techniques for studying the temporal dynamics of EXO70E2 recruitment during EXPO formation?

Several cutting-edge techniques are emerging to study the temporal dynamics of EXO70E2 recruitment during EXPO formation:

• Real-time single-molecule tracking:

  • Label EXO70E2 antibodies with quantum dots or organic fluorophores for long-term tracking

  • Implement TIRF microscopy to visualize recruitment events at the plasma membrane with high temporal resolution

  • Track single molecules to determine recruitment order and residence time

• Lattice light-sheet microscopy with adaptive optics:

  • Achieve subcellular resolution with minimal phototoxicity

  • Capture fast 3D volumes (entire cells) over extended periods

  • Track EXO70E2-positive structures throughout the entire cell volume during EXPO formation

• CRISPR-based live-cell tagging:

  • Insert fluorescent tags into endogenous EXO70E2 loci using CRISPR/Cas9

  • Maintain native expression levels and regulation

  • Validate with antibodies to confirm proper localization and function

• Microfluidic approaches with synchronized induction:

  • Develop systems to rapidly introduce inducers of EXPO formation

  • Combine with high-speed imaging to capture initial recruitment events

  • Correlate with antibody-based fixed timepoint analysis

• Förster resonance energy transfer (FRET) with temporal resolution:

  • Create FRET pairs between EXO70E2 and other exocyst components

  • Monitor interaction dynamics in real-time

  • Validate interactions through complementary antibody-based approaches

Research has established that EXO70E2 serves as a nucleation point for exocyst assembly, with several subunits (Sec5a, Sec6, Sec8, Sec10) being recruited from cytosolic pools to punctate structures when co-expressed with EXO70E2 . FRET and BiFC analyses have confirmed direct interactions between EXO70E2 and specific exocyst components like Sec6 and Sec10, suggesting a hierarchical assembly process where EXO70E2 initiates recruitment followed by sequential addition of other subunits .