Recombinant Oryza sativa subsp. japonica Formin-like protein 14 (FH14)

What is the molecular structure of Oryza sativa FH14 and how does it compare to other plant formins?

Oryza sativa formin-like proteins typically consist of three functionally distinct subdomains: an N-terminal phosphatase tensin (PTEN)-like domain, a proline-rich FH1 (Formin Homology 1) domain, and a highly conserved C-terminal FH2 (Formin Homology 2) domain . Based on structural analysis, FH14 belongs to the type II formin family, similar to Arabidopsis AFH14 which contains 18 exons and 17 introns spanning approximately 6-7 kb . The FH2 domain is particularly important as it mediates interactions with both microtubules and microfilaments, while the PTEN domain appears to direct subcellular localization .

Research comparing type I and type II formins in Physcomitrella has shown that the FH1-FH2 domains of type II formins cannot be functionally replaced by those from type I formins, suggesting evolutionary divergence between these two formin lineages .

What expression systems are most effective for producing recombinant Oryza sativa FH14?

For recombinant expression of plant formins, the following methodological approaches have proven successful:

• Bacterial expression systems: For biochemical studies, the 6-His-tagged truncated recombinant protein containing the FH1 and FH2 domains can be expressed and purified from E. coli . This approach is particularly useful for in vitro biochemical assays.

• Rice-based expression systems: Rice itself can serve as an expression platform for recombinant proteins. After successful transformation, transgenic rice cells can either be regenerated into whole plants or grown as cell cultures that can be upscaled into bioreactors . This approach maintains the native post-translational modifications.

• BY-2 cell-based expression: For studying subcellular localization and function, tobacco BY-2 cell expression systems coupled with fluorescent tags like GFP have proven effective for formin studies, as demonstrated with Arabidopsis AFH14 .

When selecting an expression system, researchers should consider whether the full-length protein or specific domains (such as FH1FH2) are needed based on experimental goals.

How can I verify the biological activity of purified recombinant FH14?

Several complementary assays can be employed to verify the biological activity of recombinant FH14:

• Actin nucleation assays: Measure the ability of purified FH14 to nucleate actin polymerization from monomeric G-actin or actin/profilin complexes using pyrene-labeled actin and fluorescence spectroscopy .

• Barbed-end capping assays: Assess FH14's ability to cap the barbed end of actin filaments, which affects filament elongation kinetics .

• Bundling assays: Examine the protein's capacity to bundle actin filaments and/or microtubules using low-speed cosedimentation assays followed by SDS-PAGE analysis .

• Microtubule-binding assays: Test direct binding to microtubules through cosedimentation assays and visualization using electron microscopy .

• Microtubule-microfilament interaction assays: For type II formins like FH14, examine whether the protein can promote interactions between microtubules and microfilaments in vitro .

These biochemical assays provide quantitative measurements of FH14's ability to function as an actin nucleator, capper, and bundler, as well as its interaction with microtubules.

What experimental approaches can reveal FH14's role in cytoskeletal organization in rice cells?

To investigate FH14's function in cytoskeletal organization, researchers can employ these methodological approaches:

• Live cell imaging with dual-labeled cytoskeleton: Express FH14-GFP in rice cells along with markers for microtubules (e.g., mCherry-TUA) and microfilaments (e.g., LifeAct-RFP) to observe dynamics in real-time .

• Oryzalin resistance assays: Test whether FH14 overexpression affects microtubule stability by treating cells with the microtubule-depolymerizing drug oryzalin. In AFH14-overexpressing cells, microtubules showed increased resistance to oryzalin, with only 52.0% of spindles and 58.2% of phragmoplasts depolymerizing compared to 75.0% and 82.0% in control cells, respectively .

• FRAP (Fluorescence Recovery After Photobleaching): Measure the dynamics of FH14 association with cytoskeletal structures in living cells.

• Immunolocalization in fixed tissues: Use specific anti-FH14 antibodies to determine precise subcellular localization at different developmental stages .

• Proximity-based labeling: Employ techniques like BioID or TurboID fused to FH14 to identify proximal proteins in vivo.

These approaches can reveal how FH14 influences the organization and dynamics of both microtubule and microfilament cytoskeletal networks in rice cells.

How do mutations in FH14/formin genes affect rice plant morphology and development?

Mutations in rice formin genes result in significant morphological abnormalities. Based on studies of the rice formin FH5/RMD (Rice Morphology Determinant):

Phenotypic consequences of formin mutations:

The rmd-1 and rmd-2 rice mutants exhibit recessive inheritance patterns, with F2 progeny showing a 3:1 segregation ratio (277 normal:83 mutant plants, χ² = 0.725, 0.1 < P < 0.5), indicating monofactorial recessive inheritance . Similar phenotypes would be expected for FH14 mutants if the protein plays comparable roles in cytoskeletal regulation.

To characterize FH14 mutant phenotypes, researchers should employ detailed morphometric analyses at multiple developmental stages combined with cellular imaging of cytoskeletal organization .

What methodologies can distinguish between FH14's effects on microtubules versus microfilaments?

To differentiate FH14's effects on different cytoskeletal components, researchers can employ these specialized approaches:

• Domain-specific mutations: Create recombinant FH14 proteins with specific mutations in the FH2 domain that selectively disrupt binding to either microtubules or microfilaments, then conduct complementation experiments in mutant plants .

• Cytoskeleton-specific drug treatments: Combine FH14 overexpression or knockdown with selective inhibitors:

  • Latrunculin B (disrupts microfilaments)

  • Oryzalin or colchicine (disrupt microtubules)

  • Analyze the differential responses to identify specific pathways .

• In vitro competitive binding assays: Determine whether FH14 shows preferential binding to microtubules or microfilaments when both cytoskeletal elements are present, using labeled proteins and microscopy or cosedimentation approaches .

• Time-resolved imaging: Examine the temporal sequence of cytoskeletal rearrangements following inducible expression or inhibition of FH14 to determine which cytoskeletal system is affected first.

Research on Arabidopsis AFH14 demonstrated that it binds directly to either microtubules or microfilaments, with the FH2 domain being essential for cytoskeleton binding and bundling. Importantly, when both cytoskeletal elements are present, AFH14 promotes interactions between them, suggesting a unique bridging function .

How can I design effective CRISPR-Cas9 experiments to create FH14 knockout or knockdown rice lines?

To generate FH14 mutant rice lines using CRISPR-Cas9, follow these methodological steps:

• Target site selection:

  • Design sgRNAs targeting conserved regions within the FH2 domain, as this domain is essential for cytoskeletal interactions

  • Select target sites with minimal off-target potential using tools like CRISPR-P or CRISPOR

  • Consider targeting multiple exons simultaneously to ensure functional knockout

• Vector construction and rice transformation:

  • Clone selected sgRNAs into a rice-optimized CRISPR-Cas9 vector

  • Transform rice calli using Agrobacterium-mediated transformation

  • Selection on hygromycin-containing media

  • Regenerate transgenic plants following established rice transformation protocols

• Mutation screening and characterization:

  • Extract genomic DNA from regenerated plantlets

  • PCR-amplify the targeted regions and sequence to identify mutations

  • Perform T7 endonuclease I assay as a rapid screening method

  • Verify protein knockdown using immunoblot analysis with anti-FH14 antibodies

• Phenotypic evaluation:

  • Examine mutant plants for morphological defects similar to those observed in rmd mutants, including growth pattern, plant height, and seed shape

  • Analyze cytoskeletal organization using immunofluorescence or live-cell imaging

• Complementation analysis:

  • Conduct genetic complementation by introducing wild-type FH14 to verify the phenotype is caused by FH14 disruption

  • Consider domain-specific complementation to determine the function of individual protein domains

When designing these experiments, remember that monofactorial recessive inheritance patterns (as seen with rmd mutants) would require analysis of segregating populations to identify homozygous mutants .

What approaches can identify potential interaction partners of FH14 in rice cells?

To identify FH14 interaction partners, employ these complementary methodological approaches:

• Immunoprecipitation coupled with mass spectrometry (IP-MS):

  • Generate tagged versions of FH14 (e.g., HA, FLAG, or GFP)

  • Perform pull-down experiments from rice cell extracts

  • Identify co-precipitated proteins using mass spectrometry

  • Validate interactions using reciprocal co-IPs and immunoblotting

• Yeast two-hybrid screening:

  • Use the full-length FH14 or specific domains (PTEN, FH1, FH2) as bait

  • Screen against a rice cDNA library

  • Validate positive interactions with directed Y2H assays and in planta methods

• Proximity-dependent labeling:

  • Fuse FH14 to BioID or TurboID biotin ligase

  • Express fusion proteins in rice cells to biotinylate proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach can reveal transient or weak interactions in native cellular environments

• In vitro reconstitution assays:

  • Test direct interactions between purified recombinant FH14 and candidate proteins

  • Examine effects on cytoskeletal dynamics or organization

  • Based on studies of AFH14, potential interactors include profilin, actin, tubulin, and other cytoskeleton-associated proteins

• FRET or BiFC analyses:

  • Create fluorescent protein fusions for FH14 and candidate interactors

  • Perform Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) in rice protoplasts or transgenic plants

  • These approaches can verify protein-protein interactions in living cells and determine subcellular locations of interactions

When analyzing potential interaction partners, particular attention should be paid to proteins involved in cytoskeletal regulation, as formin proteins like AFH14 are known to associate with both microtubule and microfilament components .

What determines the subcellular localization of FH14 in rice cells?

The subcellular localization of formin proteins is regulated by specific domains and cellular context:

• Role of the PTEN domain: In rice FH5/RMD, the N-terminal PTEN-like domain mediates localization to the chloroplast surface . Similar domain architecture in FH14 suggests the PTEN domain likely plays a crucial role in its subcellular targeting.

• Cytoskeletal association patterns: Based on studies of AFH14 in Arabidopsis, the protein localizes to microtubule-based structures including preprophase bands, spindles, and phragmoplasts during cell division . This localization is mediated by the FH2 domain, which directly binds microtubules.

• Experimental determination methods:

  • Generate FH14-GFP fusion constructs for live-cell imaging

  • Create domain deletion variants to identify localization signals

  • Perform immunolocalization with anti-FH14 antibodies in fixed cells

  • Examine co-localization with markers for different cellular compartments and cytoskeletal elements

• Microtubule dependency: Treatment with microtubule-depolymerizing drugs like oryzalin disrupts the localization of AFH14-GFP, causing the protein to either associate with irregular microtubule arrays or disperse in the cytoplasm . This suggests that intact microtubules are required for proper localization.

When studying FH14 localization, researchers should consider both cell-cycle dependent changes and possible tissue-specific variations in localization patterns, as formin distribution can vary between different cell types and developmental stages .

How can I visualize FH14 distribution during different stages of rice cell division?

To visualize FH14 during cell division, employ these methodological approaches:

• Live-cell imaging of synchronized cells:

  • Generate stable rice cell lines expressing FH14-GFP

  • Synchronize cells using aphidicolin or hydroxyurea

  • Perform time-lapse confocal microscopy during mitotic progression

  • Co-express markers for different cell cycle stages (e.g., PCNA for S phase)

• Immunofluorescence microscopy on fixed cells:

  • Collect rice root tips (for dividing cells) at defined time points

  • Fix and perform immunostaining with anti-FH14 antibodies

  • Counter-stain with anti-tubulin for mitotic structures and DAPI for chromosomes

  • Examine cells at prophase, metaphase, anaphase, and telophase/cytokinesis

• Correlative light and electron microscopy (CLEM):

  • Visualize FH14-GFP by fluorescence microscopy

  • Process the same samples for electron microscopy

  • Determine ultrastructural associations at nanometer resolution

Based on studies of AFH14 in Arabidopsis, you can expect to observe FH14 decorating:

• Preprophase bands during late G2/early prophase

• Spindle microtubules during metaphase and anaphase

• Phragmoplasts during cytokinesis

When oryzalin is applied to depolymerize microtubules, AFH14 localization is disrupted, suggesting microtubule-dependent targeting. In AFH14-overexpressing cells, both microtubules and microfilaments appear similar in length and evenly aligned, differing from the normal pattern where microfilaments form a cage around the spindle and phragmoplast microfilaments are shorter than microtubules .

What are the optimal conditions for measuring FH14's actin nucleation activity in vitro?

To accurately measure FH14's actin nucleation activity, follow these methodological guidelines:

• Protein preparation:

  • Express and purify recombinant FH14 (full-length or FH1FH2 domains) with a 6-His tag

  • Ensure protein quality through gel filtration and activity preservation by adding glycerol

  • Verify protein concentration using Bradford assay and structural integrity via circular dichroism

• Pyrene-actin polymerization assay setup:

  • Use 4-10% pyrene-labeled actin monomers (typically 2-3 μM final concentration)

  • Buffer conditions: 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM MgCl₂, 0.2 mM ATP, 0.5 mM DTT

  • Temperature: 25°C (standard) or 28°C (rice physiological temperature)

  • Measure fluorescence increase (excitation 365 nm, emission 407 nm) over time as actin polymerizes

• Profilin effect assessment:

  • Include parallel reactions with profilin (1:3 actin:profilin molar ratio)

  • Compare nucleation rates with and without profilin to assess FH14's ability to utilize profilin-actin complexes

• Controls and calibration:

  • Spontaneous polymerization control (no formin)

  • Positive control using a known formin (e.g., mDia1 FH1FH2)

  • Concentration series (50-500 nM FH14) to determine dose-response

  • Convert fluorescence units to F-actin concentration using fully polymerized samples

• Data analysis:

  • Calculate nucleation efficiency: the maximum rate of polymerization (slope of the linear phase)

  • Determine lag phase duration (time until polymerization begins)

  • Plot concentration-dependent effects of FH14 on nucleation rate

Based on similar studies with rice FH5, expect FH14 to nucleate actin polymerization from both monomeric G-actin and actin/profilin complexes, and potentially cap the barbed end of actin filaments .

How can I effectively measure FH14's influence on microtubule dynamics?

To assess FH14's effects on microtubule dynamics, implement these methodological approaches:

• In vitro reconstitution assays:

  • Microtubule binding: Perform cosedimentation assays using taxol-stabilized microtubules and recombinant FH14, analyzing bound fractions by SDS-PAGE

  • Microtubule bundling: Visualize FH14-induced microtubule bundles using negative stain electron microscopy or TIRF microscopy with fluorescent microtubules

  • Dynamic instability parameters: Measure growth/shrinkage rates and catastrophe/rescue frequencies of microtubules using TIRF microscopy with rhodamine-labeled tubulin in the presence of different FH14 concentrations

• Cell-based microtubule stability assays:

  • Generate rice cell lines with inducible FH14 expression

  • Treat with microtubule-depolymerizing drug oryzalin at 10 μM

  • Quantify resistance to depolymerization compared to control cells

  • Based on AFH14 studies, expect ~20-25% reduction in depolymerized spindles and phragmoplasts in FH14-overexpressing cells

• Microtubule recovery assays:

  • Completely depolymerize microtubules with cold treatment (4°C) or oryzalin

  • Wash out drug or return to normal temperature

  • Measure the rate and pattern of microtubule repolymerization in control versus FH14-overexpressing cells

  • Analyze using live-cell imaging or immunofluorescence

• Dual-color visualization:

  • Express FH14-GFP and RFP-tubulin in rice cells

  • Perform FRAP (Fluorescence Recovery After Photobleaching) on microtubule regions

  • Compare recovery rates in regions with high versus low FH14 localization

For data analysis, quantify:

• Percentage of intact microtubule structures after drug treatment

• Microtubule growth rates (μm/min)

• Frequency of catastrophe and rescue events

• Microtubule bundle thickness and organization

Research with AFH14 showed that its FH2 domain can directly bind to and bundle microtubules in vitro, and its overexpression increases microtubule stability against oryzalin-induced depolymerization .

What techniques can distinguish between the nucleation, elongation, and bundling activities of FH14?

To differentiate between FH14's multiple activities on cytoskeletal dynamics, employ these specialized techniques:

• Actin nucleation vs. elongation:

  • Seeded elongation assay: Add FH14 to pre-formed actin filament seeds and measure elongation rates using TIRF microscopy with labeled actin

  • Barbed-end capture: Use spectrin-actin seeds with free barbed ends to isolate elongation from nucleation

  • Capping protein competition: Add increasing concentrations of capping protein to determine if FH14 competes for barbed ends

  • Depolymerization protection: Test if FH14 prevents dilution-induced depolymerization of actin filaments, indicating barbed-end capping

• Bundling activity quantification:

  • Low-speed cosedimentation: Centrifuge at 10,000g to pellet only bundled filaments (vs. 100,000g for all filaments)

  • TIRF microscopy: Directly visualize bundle formation in real-time using fluorescently labeled cytoskeletal proteins

  • Rheology measurements: Quantify changes in viscoelastic properties of actin/microtubule networks upon FH14 addition

  • Transmission electron microscopy: Visualize and measure bundle thickness and organization

• Dual cytoskeletal regulation assessment:

  • Combined systems: Mix labeled actin and microtubules with FH14 and observe interactions using multicolor TIRF microscopy

  • Sequential addition experiments: Add FH14 first to one cytoskeletal component, then introduce the second to determine hierarchical effects

  • Domain mutation analysis: Create FH14 variants with mutations in specific FH2 residues predicted to affect either actin or microtubule binding

• Analytical approaches to distinguish activities:

  • Create a mathematical model of concentration-dependent effects for each activity

  • Fit experimental data to determine which activities predominate under different conditions

  • Use principal component analysis to identify independent activities from multiple assays

When conducting these experiments, use appropriate controls including:

• Actin or microtubules alone

• Known nucleators (Arp2/3 complex for actin)

• Known bundling proteins (α-actinin for actin, MAP2 for microtubules)

• FH2 domain alone vs. full-length protein

Based on studies of rice FH5, expect FH14 to potentially nucleate actin polymerization, cap barbed ends, and bundle both actin filaments and microtubules .

How can I conduct phylogenetic analysis to understand the evolutionary relationship of rice FH14 to other plant formins?

To perform comprehensive phylogenetic analysis of rice FH14 and related formins, follow these methodological steps:

• Sequence retrieval and curation:

  • Obtain FH14 sequences from rice subspecies (japonica, indica)

  • Collect formin sequences from diverse plant species (monocots, dicots, lower plants)

  • Include both type I and type II formins for comprehensive analysis

  • Focus on conserved domains (PTEN, FH1, FH2) for reliable alignment

• Multiple sequence alignment:

  • Align sequences using MUSCLE or MAFFT algorithms

  • Refine alignments manually, focusing on conserved regions

  • Consider domain-specific alignments (particularly the FH2 domain)

  • Generate sequence logos to visualize conservation patterns

• Phylogenetic tree construction:

  • Use maximum likelihood (RAxML, IQ-TREE) or Bayesian inference methods

  • Apply appropriate substitution models (determined by ModelTest)

  • Perform bootstrap analysis (1000 replicates) to assess branch support

  • Root the tree using formin sequences from non-plant species

• Formin classification verification:

  • Confirm FH14 clustering with type II formins (expected based on domain architecture)

  • Examine clustering patterns between rice and Arabidopsis formins

  • Previous studies show type I and type II formins form distinct evolutionary lineages with diverged functions, as domain swapping experiments revealed that the FH1-FH2 domain of type II formins cannot be functionally replaced by that of type I formins

• Selection analysis:

  • Calculate Ka/Ks ratios to identify sites under selection

  • Compare evolutionary rates between type I and type II formins

  • Identify conserved motifs that may be functionally significant

For visualization and interpretation:

• Generate a time-calibrated tree to estimate divergence times

• Correlate evolutionary patterns with known functional differences

• Map key functional residues identified in experimental studies onto the phylogeny

How does the function of rice FH14 compare to its orthologs in Arabidopsis and other model plants?

To understand functional conservation and divergence between rice FH14 and its orthologs, consider these comparative aspects:

• Structural comparison:

  Feature	Rice FH14/Formins	Arabidopsis AFH14	Other Plant Formins
  Domain architecture	PTEN-like, FH1, FH2 domains	PTEN-like, FH1, FH2 domains	Conserved domain structure in type II formins
  FH2 domain	Binds both MT and MF	Binds both MT and MF	Variable dual binding capacity
  Size	~113-114 kDa	~113.6 kDa (1033 aa)	Variable by species

• Functional conservation:

  • Cytoskeletal regulation: Both rice FH5/RMD and Arabidopsis AFH14 regulate microtubule and microfilament organization

  • Cell division roles: AFH14 localizes to preprophase bands, spindles, and phragmoplasts, suggesting conserved functions in mitosis across species

  • Biochemical activities: Similar activities including actin nucleation, filament capping, and bundling of both cytoskeletal elements

  • Subcellular targeting: The PTEN domain mediates localization in both species, though specific targets may vary (chloroplast surface for rice FH5)

• Phenotypic analysis of mutants:

  • Rice formins: Mutations in rice FH5/RMD cause bending growth, stunted plants, and abnormal seed and panicle morphology

  • Arabidopsis AFH14: Mutants show abnormal microtubule arrays during meiosis and microspore formation, affecting tetrad formation

  • Moss (Physcomitrella): Silencing type II formins leads to stunted plants with spherical cells and disrupted actin organization, while silencing type I formins produces no apparent phenotypes

• Methodological approaches for comparison:

  • Complementation experiments: Express rice FH14 in Arabidopsis afh14 mutants to test functional conservation

  • Domain swapping: Create chimeric proteins with domains from different species to identify functionally divergent regions

  • Expression pattern comparison: Compare tissue-specific and developmental expression profiles

  • Interaction partner analysis: Identify conserved and divergent binding partners

The data suggests significant functional conservation among type II formins across plant species, with these proteins playing crucial roles in cytoskeletal organization and plant morphogenesis . The inability of type I formin domains to functionally replace type II domains in moss indicates evolutionary specialization between these formin classes .

What are the most promising research directions for understanding FH14's role in rice stress responses?

Several high-potential research avenues can clarify how FH14 may function in rice stress adaptation:

• Abiotic stress response pathway integration:

  • Investigate FH14 expression patterns under drought, salinity, temperature extremes, and flooding

  • Determine whether FH14 contributes to cytoskeletal reorganization during stress adaptation

  • Examine if FH14 mutants show altered stress sensitivity compared to wild-type plants

  • Assess potential interactions with stress-responsive signaling proteins

• Methodological approaches:

  • Transcriptional profiling: Compare FH14 expression across stress conditions using RNA-seq

  • Phosphoproteomic analysis: Determine if FH14 is post-translationally modified during stress

  • Live-cell imaging: Visualize cytoskeletal dynamics in FH14-GFP plants under stress conditions

  • Field trials: Evaluate FH14 overexpression/knockdown lines under variable environmental conditions

• Cytoskeletal stress adaptation mechanisms:

  • Formins like AFH14 can increase microtubule stability against depolymerizing drugs (similar to oryzalin), reducing disruption from 75-82% to 52-58% in Arabidopsis

  • This stabilizing function could be particularly important during temperature fluctuations that disrupt cytoskeletal integrity

  • Investigate whether FH14's dual regulation of microtubules and microfilaments provides enhanced cellular resilience during stress

• Applied research potential:

  • Development of stress-tolerant rice varieties through FH14 genetic engineering

  • Identification of natural FH14 variants associated with enhanced stress tolerance

  • Creation of molecular markers for stress adaptation based on FH14 alleles

Given that cytoskeletal reorganization is a common response to various stresses, understanding FH14's role in maintaining or remodeling cytoskeletal structures could provide valuable insights into rice stress adaptation mechanisms and potential targets for crop improvement.

What novel techniques might advance our understanding of FH14's in vivo dynamics?

Emerging technologies offer powerful new approaches to study FH14 dynamics in living plants:

• Advanced microscopy methods:

  • Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy can visualize FH14-cytoskeleton interactions below the diffraction limit (~20-50 nm resolution)

  • Lattice light-sheet microscopy: Enables long-term 3D imaging with minimal phototoxicity to capture dynamic FH14 behavior over hours

  • Single-molecule tracking: Apply fluorescent protein tags or other labeling strategies to track individual FH14 molecules in living cells

  • Expansion microscopy: Physically expand cellular structures to improve resolution of FH14's interactions with cytoskeletal elements

• Optogenetic and chemogenetic approaches:

  • Develop light-inducible FH14 dimerization or activation systems to control its function with spatiotemporal precision

  • Create rapid protein degradation systems (e.g., AID or dTAG) for acute FH14 depletion

  • Implement split protein complementation with optical control to visualize specific interactions

• CRISPR-based technologies:

  • CRISPRi/CRISPRa: Target FH14 promoter regions to modulate expression levels without protein modification

  • Base editors/prime editors: Introduce specific amino acid changes to study structure-function relationships

  • CRISPR-APEX: Combine CRISPR targeting with proximity labeling to identify interaction partners at specific subcellular locations

• Computational approaches:

  • Molecular dynamics simulations of FH14-cytoskeleton interactions

  • Machine learning analysis of cytoskeletal patterns in wildtype versus FH14 mutant cells

  • Integrative modeling combining structural, biochemical, and imaging data

• In vivo biosensors:

  • Develop FRET-based sensors to monitor FH14 conformational changes during activation

  • Create tension sensors to measure forces generated during FH14-mediated cytoskeletal remodeling

  • Apply F-actin and microtubule probes to simultaneously visualize cytoskeletal dynamics and FH14 activity

These advanced techniques could reveal how FH14 dynamically interacts with and regulates both microtubule and microfilament cytoskeletal systems in living rice cells, building on fundamental insights from earlier studies of formin functions in plants .