Recombinant Oryza sativa subsp. japonica Kinesin-like calmodulin-binding protein homolog (Os04g0666900, LOC_Os04g57140), partial

What is the structural composition of Oryza sativa Kinesin-like calmodulin-binding protein?

Oryza sativa Kinesin-like calmodulin-binding protein (OsKCBP) is a C-terminal microtubule motor belonging to the Kinesin-14 family. The protein contains three unique structural domains that distinguish it from other kinesins: a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD) . The presence of both MyTH4 and talin-like domains is particularly noteworthy as these domains are typically found in some myosins but are not present in other reported kinesins outside of green plants . The protein's molecular structure reflects its evolutionary conservation within green algae and land plants, with specific adaptations that likely contribute to its specialized functions in plant cellular processes.

How evolutionarily conserved is KCBP across different species?

KCBP demonstrates remarkable evolutionary conservation specifically within green algae and land plants. Gene tree analysis has revealed that the motor domain of KCBPs belongs to a distinct clade within the Kinesin-14 family . Interestingly, comprehensive genomic analyses of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and the red alga Cyanidioschyzon merolae failed to identify KCBP homologs, suggesting that this protein is specific to the green plant lineage .

Within the plant kingdom, KCBP has been identified and isolated from diverse species including gymnosperms (Picea abies), green algae (Stichococcus bacillaris and Chlamydomonas reinhardtii), and numerous flowering plants . The conservation pattern suggests that KCBP likely emerged after the divergence of green plants from red algae and other eukaryotic lineages, indicating a specialized function in plant-specific cellular processes.

What are the key differences between OsKCBP and its homologs in other plant species?

While OsKCBP shares core structural features with other plant KCBPs, comparative analysis reveals subtle but potentially significant variations in domain organization and binding properties. In contrast to sea urchin kinesin-C (SpKinC), which contains a calmodulin-binding domain (CBD) but lacks the MyTH4 and talin-like domains, OsKCBP maintains the complete domain architecture characteristic of plant KCBPs . This suggests either a gain of these domains in the plant lineage or their loss in the animal lineage represented by SpKinC.

OsKCBP is classified as a putative microtubule-associated protein (MAP) in rice, reinforcing its role in cytoskeletal organization . When comparing OsKCBP with other plant MAP proteins such as GL7, OsCLASP, and OsMOR1, researchers can observe differences in binding partners and regulatory mechanisms that reflect species-specific adaptations in cytoskeletal dynamics.

What experimental approaches are most effective for studying OsKCBP-calmodulin interactions?

For investigating OsKCBP-calmodulin interactions, a multi-faceted experimental approach is recommended. Based on methodologies used for similar proteins, researchers should consider:

• Gel overlay assays: This technique has proven effective for demonstrating direct protein-protein interactions, as evidenced in studies of OsCBT (another calmodulin-binding protein in rice) . The assay can visualize the binding of calmodulin to OsKCBP under various calcium concentrations.

• Gel mobility shift assays: These assays help characterize the binding properties and can determine whether the interaction is calcium-dependent or calcium-independent .

• Site-directed mutagenesis: This approach is crucial for identifying specific amino acid residues involved in the interaction. For OsKCBP, focus on the two types of calmodulin-binding domains that have been identified in similar proteins: the IQ motif (calcium-independent) and the calcium-dependent motif .

• Co-expression systems: Utilizing dual-transgene vectors expressing both OsKCBP and calmodulin simultaneously provides valuable insights into their interaction dynamics in vivo . The recently developed p35S::GFP-α-tubulin-p35S::mCherry vector system can be adapted for this purpose, allowing visualization of the spatial and temporal aspects of the interaction.

How can researchers effectively design experiments to study the dual functionality of OsKCBP in microtubule binding and calmodulin interaction?

Designing experiments to investigate the dual functionality of OsKCBP requires integrated approaches that simultaneously monitor both microtubule binding and calmodulin interaction. An effective experimental design should include:

• Dual fluorescent tagging: Utilize the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry) to simultaneously visualize OsKCBP and either microtubules or calmodulin in living cells . This allows for real-time observation of dynamic interactions.

• Domain deletion/mutation analysis: Create a series of OsKCBP constructs with specific domains deleted or mutated to dissect the contributions of different regions to microtubule binding versus calmodulin interaction.

• Calcium concentration manipulation: Since calmodulin binding often depends on calcium concentration, experiments should include controlled variations in calcium levels to determine how this affects the dual functionality of OsKCBP.

• Comparative analysis with other MAPs: Include parallel experiments with other rice MAPs (such as GL7, OsCLASP, and OsMOR1) to contextualize OsKCBP's unique properties .

What methodological approaches can resolve contradictory data regarding OsKCBP regulation by calcium/calmodulin?

When faced with contradictory data regarding OsKCBP regulation by calcium/calmodulin, researchers should implement the following methodological approaches:

• Standardized biochemical conditions: Establish consistent experimental conditions across different studies, particularly regarding calcium concentrations, pH, and ionic strength. Studies on OsCBT have demonstrated that slight variations in these parameters can significantly affect calmodulin binding .

• High-resolution structural analysis: Employ techniques such as X-ray crystallography or cryo-electron microscopy to determine the precise structural changes that occur in OsKCBP upon calcium/calmodulin binding. This can help resolve discrepancies in functional data.

• Live-cell imaging with calcium indicators: Combine fluorescently tagged OsKCBP with calcium indicators to correlate calcium fluctuations with changes in OsKCBP localization and activity in real time.

• Comparative analysis across species: Analyze KCBP regulation in multiple plant species, including both monocots and dicots, to determine whether contradictory results reflect species-specific differences or experimental artifacts.

• Quantitative binding assays: Use surface plasmon resonance or isothermal titration calorimetry to precisely measure binding affinities under various conditions, providing quantitative data to resolve qualitative discrepancies.

What are the optimal conditions for expressing recombinant OsKCBP in heterologous systems?

For optimal expression of recombinant OsKCBP in heterologous systems, researchers should consider the following conditions:

• Vector selection: The dual-transgene expression vector system (p35S::GFP-α-tubulin-p35S::mCherry) has proven effective for expressing rice MAP proteins, including OsKCBP . This system allows for co-expression with tubulin, which may enhance proper folding and functionality.

• Expression system: Tobacco (Nicotiana benthamiana) leaf transient expression system has been successfully used for rice MAPs . This plant-based expression system provides a more natural cellular environment for plant proteins compared to bacterial or yeast systems.

• Transformation method: Agrobacterium-mediated transformation is recommended, as it has been validated for the expression of rice MAPs including OsKCBP .

• RNA extraction and cDNA synthesis protocol:

  • Extract total RNA using the RNeasy Plant Mini Kit

  • Use approximately 0.1g of fresh rice young inflorescence tissue

  • Grind tissue to powder in liquid nitrogen

  • Follow the RNeasy Mini Handbook protocol with RNase-free equipment

  • For reverse transcription, use 1μg total RNA with the SuperScript III First-Strand Synthesis System

• PCR amplification conditions:

  • Use high-fidelity polymerase such as KOD-Plus-Neo

  • Include appropriate PCR buffer, 25mM MgSO₄, and 2mM dNTPs

  • Design primers with appropriate restriction sites or overlaps for Gibson assembly

What purification methods yield the highest activity retention for recombinant OsKCBP?

To achieve maximum activity retention during purification of recombinant OsKCBP, a strategic multi-step approach is recommended:

• Initial extraction: For plant tissue-expressed OsKCBP, use a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 10% glycerol, 1mM DTT, and protease inhibitor cocktail. This composition preserves protein structure while preventing proteolytic degradation.

• Affinity chromatography: If working with tagged OsKCBP (e.g., His-tagged or GST-fusion), utilize the corresponding affinity resin. For calmodulin-binding proteins like OsKCBP, calmodulin-agarose chromatography has proven effective in isolating functional proteins with intact calmodulin-binding domains .

• Ion exchange chromatography: As a secondary purification step, anion exchange using a gradient of 0-500mM NaCl can separate OsKCBP from contaminating proteins with different charge properties.

• Activity preservation considerations:

  • Maintain 1-5mM calcium or EGTA (depending on whether studying calcium-dependent or independent functions)

  • Include 10% glycerol in all buffers to prevent protein denaturation

  • Keep temperatures at 4°C throughout purification

  • Consider adding microtubule stabilizers if microtubule-binding activity is to be preserved

• Quality control assessments:

  • SDS-PAGE to verify purity

  • Western blotting with anti-KCBP antibodies

  • Microtubule co-sedimentation assays to confirm activity retention

  • Calmodulin binding assays (gel overlay) to confirm functional CBD

How can researchers effectively design primers for cloning OsKCBP for different experimental applications?

Designing effective primers for cloning OsKCBP requires careful consideration of multiple factors to ensure successful amplification and subsequent experimental applications:

• Basic primer design principles:

  • Maintain optimal primer length (18-30 nucleotides)

  • Ensure GC content between 40-60%

  • Check melting temperatures (Tm) with aim for 55-65°C

  • Avoid secondary structures and primer-dimers

• Application-specific modifications:

  • For directional cloning into expression vectors, add appropriate restriction sites with 3-6 nucleotide overhangs

  • For Gibson assembly, include 15-25bp overlaps with adjacent fragments

  • For Gateway cloning, incorporate attB sites

• Domain-specific considerations:
  When studying specific domains of OsKCBP, design primers that precisely target domain boundaries:

  Domain	Approximate Position	Primer Design Considerations
  Motor domain	N-terminal	Include start codon; avoid truncating conserved motifs
  MyTH4	Middle region	Check secondary structure predictions to avoid disruption
  Talin-like	Middle region	Ensure complete domain capture
  CBD	C-terminal	Include both IQ motif and Ca²⁺-dependent motifs

• For fluorescent protein fusions:

  • When creating GFP or mCherry fusions, design primers that maintain the reading frame between OsKCBP and the fluorescent tag

  • Consider adding a flexible linker sequence (e.g., GSGSGS) to prevent steric hindrance

• Recommended primer examples for full-length OsKCBP:

  • Forward primer for N-terminal tagging:
    5'-NNNNCCATGGATGNNNNNNNNNNNNNNNN-3'
    (where ATG is the start codon and CCATGG is an NcoI site)

  • Reverse primer for C-terminal tagging:
    5'-NNNNGCGGCCGCTTANNNNNNNNNNNNNNNN-3'
    (where TTA is a reverse complement of the stop codon and GCGGCCGC is a NotI site)

How does OsKCBP function compare to other calmodulin-binding proteins in rice development?

OsKCBP functions in rice development can be contrasted with other calmodulin-binding proteins such as OsCBT (Oryza sativa CaM-binding transcription factor) to reveal distinct regulatory mechanisms:

• Functional domains comparison:
  While OsKCBP combines motor protein functionality with calmodulin regulation, OsCBT functions primarily as a transcription factor containing a CG-1 homology DNA-binding domain, three ankyrin repeats, a transcriptional activation domain, and five calmodulin-binding motifs . This fundamental difference positions OsKCBP in cytoskeletal dynamics while OsCBT operates in transcriptional regulation.

• Calmodulin binding mechanisms:
  Both proteins exhibit dual calmodulin-binding capabilities. OsCBT contains two different types of functional CaM-binding domains: an IQ motif (calcium-independent) and a calcium-dependent motif . Similarly, OsKCBP likely possesses both binding modes, though the regulatory consequences differ based on their distinct cellular functions.

• Developmental significance:
  OsKCBP likely influences cellular architecture, division plane orientation, and intercellular transport through its microtubule-binding properties. In contrast, OsCBT regulates gene expression by binding to specific DNA sequences (5'-TWCG(C/T)GTKKKKTKCG-3') and activating reporter gene expression . This dichotomy represents complementary mechanisms by which calcium/calmodulin signaling orchestrates rice development through both cytoskeletal and transcriptional pathways.

• Regulatory dynamics:
  Intriguingly, while OsCBT-mediated transcriptional activation is inhibited by calmodulin co-expression , OsKCBP activity may be either enhanced or inhibited by calmodulin binding depending on calcium concentration. This suggests that the calcium/calmodulin signaling system can exert both positive and negative regulatory effects on different cellular processes simultaneously.

What are the implications of OsKCBP expression patterns for functional rice breeding programs?

Understanding OsKCBP expression patterns has significant implications for rice breeding programs focused on developing functional rice varieties:

• Correlation with developmental traits:
  OsKCBP's role in cytoskeletal organization may influence key developmental processes that affect agronomically important traits. Breeding programs targeting improved yield potential, particularly those focusing on embryo size enhancement (such as giant embryo rice varieties like Tainung78), should consider OsKCBP expression patterns as potential markers or targets .

• Integration with marker-assisted selection (MAS):
  Current high-yield functional rice breeding employs marker-assisted selection for genes like GE (giant embryo) and OsALDH7 (aldehyde dehydrogenase, golden-like endosperm) . Given OsKCBP's potential involvement in cellular architecture, analyzing correlations between OsKCBP expression/variants and these established markers could enhance breeding efficiency.

• Application in diverse rice varieties:
  The development of functional rice varieties, including colored rice with enhanced nutritional profiles (such as purple waxy rice CNY922401 and red waxy rice TNGSW26), could benefit from OsKCBP expression analysis . Different expression patterns or allelic variants of OsKCBP might correlate with specific grain quality traits or stress responses.

• Technical considerations for breeding applications:
  When incorporating OsKCBP analysis into breeding programs, researchers should consider:

  • Developing PCR-based markers for specific OsKCBP alleles

  • Creating expression analysis platforms for measuring OsKCBP levels in developing tissues

  • Evaluating OsKCBP expression under different environmental conditions to assess stability

How can comparative analysis of OsKCBP with KCBPs from other plant species inform evolutionary understanding of cytoskeletal regulation?

Comparative analysis of OsKCBP with KCBPs from other plant species provides valuable insights into the evolution of cytoskeletal regulation in plants:

• Evolutionary trajectory reconstruction:
  The presence of KCBP specifically in green algae and land plants, but not in red algae or non-plant eukaryotes, suggests a gain of this specialized motor protein after the divergence of green plants . This represents a key innovation in plant cytoskeletal evolution that may have facilitated adaptations to terrestrial environments.

• Domain acquisition patterns:
  Comparative analysis reveals that the MyTH4 and talin-like domains characteristic of plant KCBPs are absent in sea urchin kinesin-C (SpKinC), despite the latter containing a calmodulin-binding domain . This suggests either domain acquisition in the plant lineage or domain loss in the animal lineage, with important implications for understanding the evolutionary forces shaping cytoskeletal proteins.

• Functional specialization across plant taxa:
  By comparing OsKCBP with KCBPs from diverse plants including gymnosperms (Picea abies), green algae (Stichococcus bacillaris, Chlamydomonas reinhardtii), and various flowering plants , researchers can trace how functional specialization may have occurred during plant evolution. This provides a molecular framework for understanding the diversification of plant morphology and development.

• Methodological approach for evolutionary analysis:
  To conduct rigorous comparative studies, researchers should:

  • Perform comprehensive phylogenetic analyses using both maximum likelihood and Bayesian methods

  • Analyze selection pressures on different domains using dN/dS ratios

  • Conduct synteny analysis to identify genomic rearrangements affecting KCBP genes

  • Use ancestral sequence reconstruction to infer the properties of progenitor KCBP proteins

These evolutionary insights not only enhance our fundamental understanding of plant cytoskeletal evolution but also inform applied research by identifying conserved functional elements that might be targeted in crop improvement programs.

What are common challenges in purifying active recombinant OsKCBP and how can they be addressed?

Researchers frequently encounter several challenges when purifying active recombinant OsKCBP. These issues and their solutions include:

• Protein degradation during extraction:

  • Problem: OsKCBP's large size (predicted 103-kDa) makes it susceptible to proteolytic degradation

  • Solution: Include a comprehensive protease inhibitor cocktail in extraction buffers, maintain low temperatures throughout purification, and consider adding stabilizing agents like glycerol (10-15%)

• Insolubility when expressed in heterologous systems:

  • Problem: Motor proteins often form inclusion bodies when overexpressed

  • Solution: Optimize expression conditions by reducing temperature (16-20°C), using slower induction protocols, or considering expression as separate functional domains

• Loss of calmodulin-binding activity:

  • Problem: The calmodulin-binding domain may become inaccessible during purification

  • Solution: Include calcium (1-5 mM) or EGTA in purification buffers depending on the binding mode being studied, and validate activity using gel overlay assays with calmodulin-HRP probes

• Decreased microtubule-binding function:

  • Problem: The motor domain may lose activity during purification

  • Solution: Include ATP/ADP in buffers at appropriate concentrations, add stabilizing agents like glycerol, and verify activity using microtubule co-sedimentation assays

• Co-purification of contaminants:

  • Problem: Bacterial proteins with similar properties may co-purify

  • Solution: Implement a multi-step purification strategy combining affinity chromatography with ion exchange and/or size exclusion chromatography

How should researchers interpret conflicting localization data for OsKCBP in different cell types?

When confronted with conflicting localization data for OsKCBP across different cell types, researchers should adopt a structured analytical approach:

• Systematic comparison of experimental conditions:
  Document and compare all variables between conflicting studies, including:

  • Cell type-specific factors (differentiation state, metabolic activity)

  • Visualization methods (antibody detection vs. fluorescent protein fusion)

  • Fixation protocols (which can affect microtubule and associated protein preservation)

  • Growth conditions and developmental stage

• Validation using multiple approaches:

  • Compare immunolocalization with GFP fusion proteins

  • Use both N-terminal and C-terminal tags to identify potential interference with localization signals

  • Apply the dual-transgene expression system to simultaneously visualize OsKCBP with both tubulin and calmodulin

• Temporal analysis:

  • Conduct time-course studies to determine if localization varies throughout the cell cycle

  • Consider calcium flux changes that might affect calmodulin binding and subsequent localization

• Quantitative image analysis:

  • Implement standardized quantification methods to assess the degree of colocalization with various cellular structures

  • Use statistical approaches to determine significance of observed differences

• Functional correlation:

  • Test whether localization differences correlate with functional differences using activity assays

  • Consider whether post-translational modifications might explain cell type-specific localization patterns

What strategies can overcome challenges in analyzing OsKCBP-microtubule interactions in vivo?

Analyzing OsKCBP-microtubule interactions in living cells presents unique challenges that can be addressed through specialized approaches:

• Optimized live-cell imaging:

  • Utilize the dual-transgene expression vector system (p35S::GFP-α-tubulin-p35S::mCherry-OsKCBP) for simultaneous visualization

  • Employ spinning disk confocal microscopy or light sheet microscopy to reduce photobleaching and phototoxicity

  • Use environmental chambers to maintain optimal growth conditions during imaging

• Sensitivity to calcium fluctuations:

  • Co-express calcium indicators (e.g., R-GECO) alongside fluorescently tagged OsKCBP to correlate calcium levels with interaction dynamics

  • Implement calcium uncaging techniques to precisely manipulate local calcium concentrations while observing effects on OsKCBP-microtubule binding

• Addressing photobleaching:

  • Utilize new fluorescent protein variants with enhanced photostability

  • Implement computational approaches like denoising algorithms to extract data from lower exposure images

  • Consider using photoconvertible fluorescent proteins for pulse-chase experiments

• Quantification challenges:

  • Develop automated image analysis workflows using machine learning approaches to quantify colocalization

  • Implement Fluorescence Recovery After Photobleaching (FRAP) or Fluorescence Loss In Photobleaching (FLIP) to assess binding kinetics

  • Use single-molecule tracking to characterize individual OsKCBP-microtubule interactions

• Tissue penetration limitations:

  • For studies in intact plant tissues, consider using clearing techniques compatible with fluorescent proteins

  • Employ multi-photon microscopy for deeper tissue penetration

  • Develop tissue-specific expression systems to target analysis to particular cell types of interest

By implementing these advanced strategies, researchers can overcome the technical challenges inherent in studying dynamic protein-cytoskeleton interactions in living plant cells, leading to more accurate characterization of OsKCBP function in its native context.