Recombinant Arabidopsis thaliana WPP domain-interacting protein 2 (WIP2)

What is the biological role of WIP2 in Arabidopsis thaliana?

WIP2 is essential for nuclear elongation in epidermal cell types such as root hairs and trichomes. It forms a complex with SUN1/2 proteins at the nuclear envelope (NE), serving as a scaffold to recruit WIT2 and myosin XI-i, which link the nucleoskeleton to the cytoskeleton . Methodological verification involves:

• Mutant phenotyping: Compare nuclear shape in wip2 mutants (e.g., invaginated nuclei) versus wild-type using confocal microscopy .

• Localization studies: Fluorescently tag WIP2 to track its NE localization via transient expression in protoplasts .

• Interaction assays: Use yeast two-hybrid systems to confirm binding between WIP2 and SUN1/2 .

How is recombinant WIP2 typically expressed and purified for experimental use?

Recombinant WIP2 is often expressed in E. coli or plant-based systems. Key steps include:

• Cloning: Amplify the WIP2 coding sequence (CDS) with a His-tag using primers designed for Gateway-compatible vectors .

• Expression: Use BL21(DE3) cells induced with 0.5 mM IPTG at 18°C for 16 hours to minimize inclusion bodies.

• Purification: Employ nickel-NTA affinity chromatography followed by size-exclusion chromatography to isolate monomeric WIP2 .

• Validation: Confirm purity via SDS-PAGE and identity through Western blotting with anti-His antibodies .

What experimental techniques are used to study WIP2-protein interactions?

• Co-immunoprecipitation (Co-IP): Transiently co-express WIP2-GFP and SUN2-mCherry in Nicotiana benthamiana, immunoprecipitate with GFP-Trap beads, and detect partners via immunoblotting .

• Bimolecular fluorescence complementation (BiFC): Split YFP fragments fused to WIP2 and WIT2 are co-expressed to visualize interaction sites .

• Tandem affinity purification (TAP): Use transgenic Arabidopsis lines expressing TAP-tagged WIP2 to isolate native complexes for mass spectrometry .

How does WIP2 contribute to nuclear morphology in plant cells?

WIP2 stabilizes SUN-WIT2-myosin XI-i bridges at the NE, enabling cytoskeletal forces to elongate nuclei. CRWN1 independently shapes nuclei via nucleoplasmic filaments, creating a dual-mechanism model . Investigate this using:

• Live-cell imaging: Track nuclear shape changes in wip2 mutants expressing GFP-tagged histone H2B.

• Quantitative morphology analysis: Measure nuclear aspect ratio (length/width) in ImageJ across 100+ cells per genotype .

How can researchers resolve contradictions in WIP2’s role across different cell types?

WIP2 exhibits functional divergence in trichomes versus root hairs due to paralog-specific interactions. Strategies include:

• Cell type-specific silencing: Use Cellulose Synthase 6 (CESA6) or GLABRA2 promoters to knock down WIP2 in specific tissues .

• Single-cell RNA-seq: Profile WIP2 interaction partners (e.g., SUN1, WIT2) across cell types to identify context-dependent networks .

• Phenotypic benchmarking: Compare nuclear invagination depth in wip2 trichomes (severe) versus root hairs (mild) using 3D reconstructions .

What are the challenges in characterizing post-translational modifications (PTMs) of recombinant WIP2?

PTMs like phosphorylation or SUMOylation regulate WIP2’s NE localization and interactions. Address this via:

• Phosphoproteomics: Treat purified WIP2 with lambda phosphatase and analyze shifts in electrophoretic mobility .

• SUMOylation assays: Co-express WIP2 with SUMO E3 ligase AtSIZ1 in Arabidopsis protoplasts, immunoprecipitate, and detect SUMO conjugates using anti-SUMO1 antibodies .

• In vitro modification: Incubate recombinant WIP2 with kinase extracts from Arabidopsis roots and perform LC-MS/MS to identify modification sites .

How do genetic backgrounds affect WIP2 function in mutant complementation studies?

WIP2 mutant phenotypes vary depending on ecotype (e.g., Col-0 vs. Ler). Standardize experiments by:

• Backcrossing: Introgress wip2 mutations into uniform backgrounds for ≥3 generations.

• Phenotypic scoring: Use quantitative metrics (e.g., nuclear aspect ratio) rather than binary classifications .

• Transcriptional profiling: Compare WIP2 expression levels in complemented lines via qRT-PCR with UBQ10 as a reference .

What strategies optimize WIP2 interaction studies in dynamic cellular environments?

• FRAP (Fluorescence Recovery After Photobleaching): Measure WIP2-GFP mobility at the NE under cytoskeletal disruption (e.g., latrunculin B treatment) .

• Crosslinking: Treat cells with 1% formaldehyde before Co-IP to capture transient interactions .

• Computational modeling: Predict WIP2 binding hotspots using AlphaFold2-predicted structures and dock with SUN1/2 .

Data Tables

Table 1: WIP2 Interaction Partners and Functional Roles

Table 2: Common Experimental Issues and Solutions