Recombinant Xenopus laevis Ciliogenesis and planar polarity effector 2 (cplane2)

What is the function of CPLANE2 in Xenopus laevis development?

CPLANE2 (also known as RSG1) is a critical component of the Ciliogenesis and PLANar polarity Effector (CPLANE) complex in Xenopus laevis. It functions as a small GTPase that is essential for ciliogenesis. Initially identified as a Fuz-interacting protein, CPLANE2 plays a vital role in proper cilia formation and function . Recent research indicates that CPLANE2 acts at a relatively late step in ciliogenesis and is particularly important for basal body docking .

The CPLANE complex, which includes CPLANE2, is a multiprotein complex that controls both planar cell polarity and ciliogenesis in vertebrates. In Xenopus specifically, disruption of CPLANE2 leads to defects in basal body docking and subsequent ciliogenesis issues, highlighting its developmental importance .

How does CPLANE2 relate to the broader CPLANE complex?

CPLANE2 is one of five proteins that constitute the vertebrate CPLANE complex, which is essential for ciliogenesis. The complex includes:

• JBTS17/CPLANE1

• RSG1/CPLANE2

• Fuz/CPLANE3

• Intu/CPLANE4

• Wdpcp/CPLANE5

Within this complex, CPLANE2 appears to be the most downstream component in the assembly hierarchy . Unlike other components (Fuz and Intu) that function together as a GEF for Rab23, CPLANE2 is suggested to act as an effector rather than a substrate of the Intu/Fuz GEF . The complex assembles hierarchically near basal bodies, with CPLANE2 being recruited last in the sequence, which underscores its function at a late step in ciliogenesis .

What experimental methods are commonly used to study CPLANE2 in Xenopus?

Research on CPLANE2 in Xenopus typically employs several methodological approaches:

• Morpholino knockdown: Antisense morpholino oligonucleotides are used to knock down CPLANE2 expression to observe resulting phenotypes .

• mRNA rescue experiments: Wild-type or variant CPLANE2 mRNA is co-injected with knockdown reagents to assess functional rescue of phenotypes, as demonstrated in basal body docking experiments .

• Confocal microscopy: To visualize basal body docking defects in multiciliated cells (MCCs), researchers use this technique to measure basal body depth and distribution .

• Protein interaction studies: To characterize CPLANE2's interactions with other CPLANE complex members, particularly its interaction with Fuz .

• Structural analysis: Combining experimental data with computational predictions (like AlphaFold3) to understand protein structure-function relationships .

How do pathogenic variants in CPLANE2 affect its molecular function and cause ciliopathies?

Pathogenic variants in CPLANE2 have been linked to human ciliopathies with phenotypes resembling Oral-Facial-Digital syndrome (OFD) . Molecular analysis of these variants reveals distinct mechanisms of dysfunction:

• Structural disruption: The A76P variant (equivalent to A76P in human/S72P in Xenopus) anchors the α1 helix, and substitution with proline severely disrupts helix formation, compromising protein stability and function .

• GTP binding interference: The G118E variant (G114E in Xenopus) resides within the G3 region of the GTP binding pocket, adjacent to E119, which is critical for GTP binding. This mutation likely impairs GTP binding and subsequent activity .

• Interface perturbation: The R188W variant (D184W in Xenopus) is positioned on the outer edge of the α4 helix, away from both the GTP-binding region and known Fuz interaction sites, suggesting it may disrupt other molecular interactions .

Functional studies in Xenopus MCCs demonstrated that these variants fail to fully rescue basal body docking defects caused by CPLANE2 knockdown, with the S72P variant showing the most severe impairment and D184W exhibiting partial rescue capability .

What is the relationship between CPLANE2's GTPase activity and its function in basal body docking?

CPLANE2's GTPase activity is intimately linked to its function in basal body docking through a complex molecular mechanism:

• CPLANE2 is a small GTPase that binds GTP in vitro . The canonical T→N substitution in the G1 region of GTPases, which typically reduces GTP binding and hydrolysis, impairs CPLANE2 function .

• In Xenopus multiciliated cells (MCCs), CPLANE2 knockdown disrupts basal body docking, increasing the distance between basal bodies and the apical surface . This defect can be rescued by wild-type CPLANE2 but not by variants with impaired GTP binding (such as the G114E variant) .

• Interestingly, variants with different molecular defects show varying abilities to rescue basal body docking. The S72P variant (disrupting protein structure) completely fails to rescue, while G114E (affecting GTP binding) provides modest rescue, and D184W (potentially affecting protein interactions) offers significant but incomplete rescue .

This differential rescue capacity suggests that while GTP binding is important for CPLANE2 function, the protein likely has multiple functional domains contributing to basal body docking through distinct mechanisms.

How does CPLANE2 coordinate with IFT complexes during ciliogenesis?

The relationship between CPLANE2 and Intraflagellar Transport (IFT) complexes represents a critical interface in ciliogenesis regulation:

• The CPLANE complex, including CPLANE2, is implicated in the recruitment of IFT-A2 proteins to the base of cilia . In Xenopus, Fuz and Intu (other CPLANE components) are essential for this recruitment process .

• While CPLANE2 acts at a relatively late step in ciliogenesis compared to other CPLANE components, its function appears to be upstream of proper IFT complex localization and function .

• Pathogenic variants in CPLANE2 and other CPLANE components disrupt both lipid binding by the CPLANE complex and IFT-A2 recruitment to basal bodies , suggesting a mechanistic link between these processes.

• The hierarchical assembly of the CPLANE complex, with CPLANE2 as the most downstream component, places it in a strategic position to potentially serve as an effector that directly interfaces with the IFT machinery after the CPLANE complex is fully assembled .

This coordination appears essential for proper cilia formation, as disruption of CPLANE2 leads to ciliogenesis defects that resemble those seen with IFT complex dysfunction.

What are the precise molecular mechanisms by which CPLANE2 facilitates basal body docking?

The molecular mechanisms underlying CPLANE2's role in basal body docking involve several coordinated processes:

• CPLANE2, as part of the CPLANE complex, localizes near basal bodies where it participates in the hierarchical assembly of the complex .

• GTP binding appears critical for CPLANE2's function, as variants affecting the GTP binding pocket (G114E) show impaired ability to rescue basal body docking defects .

• CPLANE2 likely acts as an effector downstream of the Fuz/Intu GEF activity, which activates Rab23. This pathway has been implicated in the docking of basal bodies to the apical surface .

• Experimental evidence in Xenopus MCCs shows that knockdown of CPLANE2 increases the distance between basal bodies and the apical surface, demonstrating its direct involvement in the docking process .

• The observation that different pathogenic variants of CPLANE2 show varying abilities to rescue basal body docking defects suggests multiple functional domains contribute to this process through distinct mechanisms .

These findings collectively indicate that CPLANE2 serves as a critical effector in translating upstream signals from the CPLANE complex to the basal body docking machinery, potentially through interactions with cytoskeletal elements or membrane components that remain to be fully characterized.

What are the key considerations when designing CPLANE2 knockdown and rescue experiments in Xenopus?

When designing CPLANE2 knockdown and rescue experiments in Xenopus, researchers should consider:

• Knockdown approach selection:

  • Morpholino oligonucleotides have been successfully used to target CPLANE2

  • CRISPR-Cas9 approaches may provide more specific gene targeting

  • Consider controls for off-target effects, including rescue experiments

• Rescue construct design:

  • Use species-specific sequences that are resistant to knockdown (for morpholinos)

  • Include appropriate tags (GFP, FLAG) that don't interfere with protein function

  • For structure-function analysis, create targeted mutations based on conserved domains and pathogenic human variants

• Developmental timing:

  • CPLANE2 is present throughout early Xenopus development

  • Consider stage-specific effects, particularly around the midblastula transition (MBT) when checkpoint control changes

  • For ciliogenesis studies, target interventions to stages preceding multiciliated cell differentiation

• Quantification methods:

  • For basal body docking, measure the distance between basal bodies and the apical surface using confocal microscopy

  • Use multiple markers to visualize both basal bodies and the apical membrane

  • Analyze sufficient numbers of cells across multiple embryos for statistical significance

• Appropriate controls:

  • Include wild-type rescue, catalytically inactive mutants, and empty vector controls

  • Use human disease variants to establish translational relevance

How can researchers effectively analyze CPLANE2's role in the molecular hierarchy of ciliogenesis?

To effectively analyze CPLANE2's position in the molecular hierarchy of ciliogenesis:

• Sequential depletion and epistasis experiments:

  • Knockdown CPLANE2 together with other CPLANE components or ciliary proteins

  • Assess rescue capabilities of upstream vs. downstream components

  • Use double knockdown/overexpression approaches to establish hierarchical relationships

• Temporal analysis of protein recruitment:

  • Perform time-lapse imaging of fluorescently tagged CPLANE2 and other components

  • Quantify the sequence of protein recruitment to forming cilia

  • Correlate with morphological stages of ciliogenesis

• Interaction mapping:

  • Use proximity labeling (BioID/TurboID) to identify proteins in the vicinity of CPLANE2

  • Perform co-immunoprecipitation followed by mass spectrometry to identify interacting partners

  • Map interaction domains through truncation analysis

• Functional assays at distinct steps of ciliogenesis:

  • Examine basal body migration, docking, axoneme extension, and ciliary membrane formation separately

  • Use specific markers for each process to isolate CPLANE2's effects

  • Quantify effects on IFT dynamics using particle tracking

• Comparative analysis across species:

  • Compare CPLANE2 function in Xenopus with orthologs in mice and human systems

  • Use conservation analysis to identify functionally critical domains

  • Correlate evolutionary conservation with functional importance in the ciliogenesis hierarchy

What techniques can be used to study the GTPase activity of CPLANE2 and its relevance to ciliogenesis?

Several complementary techniques can be employed to study CPLANE2's GTPase activity:

• In vitro GTPase assays:

  • Express and purify recombinant CPLANE2 protein

  • Measure GTP binding using fluorescent GTP analogs or radiolabeled GTP

  • Quantify GTP hydrolysis rates using malachite green phosphate assays

  • Compare wild-type activity with disease-associated variants

• Structure-function analysis:

  • Design mutations in key GTPase domains (G1-G5) based on structural predictions

  • Test canonical mutations that affect binding (T→N) or hydrolysis (Q→L) in rescue assays

  • Use AlphaFold3 or similar tools to predict structural impacts of mutations

• Live GTPase activity sensors:

  • Develop FRET-based sensors to monitor CPLANE2 GTPase activity in vivo

  • Use GTP-locked or GDP-locked mutants as controls

  • Track activity dynamics during ciliogenesis stages

• Visualization of GTP-bound state:

  • Use conformation-specific antibodies to detect active CPLANE2

  • Apply proximity ligation assays to identify interactions that depend on GTP-bound state

  • Employ GTP-binding domain pull-downs to isolate active CPLANE2

• Correlation with ciliogenesis phenotypes:

  • Compare ciliogenesis defects caused by GTPase-deficient mutants versus protein-null conditions

  • Assess basal body docking specifically with mutants that affect GTP binding versus other domains

  • Test whether artificially activating downstream effectors can bypass GTPase-deficient CPLANE2

How should researchers interpret contradictory findings about CPLANE2 function across different model systems?

When facing contradictory findings about CPLANE2 across model systems, researchers should:

• Consider developmental context differences:

  • Xenopus embryos undergo significant developmental changes at the midblastula transition (MBT) that affect checkpoint engagement and cell cycle regulation

  • Compare experimental timepoints relative to developmental milestones across species

  • Assess whether contradictions reflect genuine biological differences or experimental variations

• Evaluate protein conservation and divergence:

  • Analyze sequence conservation of CPLANE2 across species, particularly in functional domains

  • Consider whether splice variants or posttranslational modifications differ between systems

  • Examine if interaction partners are conserved across the studied species

• Compare experimental approaches systematically:

  • Evaluate differences in knockout/knockdown efficiency and specificity

  • Consider differences between acute (morpholino) versus chronic (genetic) loss of function

  • Assess whether cellular contexts (embryonic versus cultured cells) explain differences

• Integrate findings with evolutionary perspective:

  • Consider whether differences reflect evolutionary specialization of ciliary functions

  • Determine if contradictions align with known cilia type-specific roles (motile versus primary)

  • Evaluate whether contradictions reflect fundamental differences in ciliogenesis regulation

• Design reconciling experiments:

  • Cross-validate findings using identical reagents across systems when possible

  • Perform rescue experiments with orthologs from different species

  • Use domain swapping to identify regions responsible for species-specific functions

What are the key considerations when analyzing the role of CPLANE2 in human ciliopathies based on Xenopus models?

When translating findings from Xenopus CPLANE2 studies to human ciliopathies:

• Validate variant effects in both systems:

  • Test human disease variants in Xenopus models for functional conservation

  • Compare phenotypic effects of equivalent mutations (e.g., human A76P vs. Xenopus S72P)

  • Consider species-specific differences in protein structure and function

• Compare tissue-specific effects:

  • Analyze whether ciliopathy phenotypes correlate with tissue-specific expression patterns

  • Consider differences in cilia-dependent development between species

  • Evaluate whether phenotypic differences reflect species-specific developmental programs

• Establish phenotype-genotype correlations:

  • Categorize mutations based on molecular mechanisms (GTP binding, protein stability, etc.)

  • Correlate different mutation types with specific ciliopathy features

  • Use quantitative phenotyping to detect subtle functional differences between variants

• Consider evolutionary conservation of mechanisms:

  • Assess whether basal body docking mechanisms are conserved between Xenopus and humans

  • Evaluate if the CPLANE complex composition and assembly are maintained across species

  • Determine whether downstream effectors are conserved between models

• Integrate data from multiple models:

  • Cross-validate findings using cell culture, organoid, and animal models

  • Consider whether differences reflect experimental limitations or biological divergence

  • Use complementary approaches to overcome limitations of individual model systems

How can researchers distinguish between direct and indirect effects of CPLANE2 disruption on ciliogenesis?

Distinguishing direct from indirect effects of CPLANE2 disruption requires:

• Temporal analysis of defects:

  • Perform time-course experiments to determine the sequence of defects following CPLANE2 disruption

  • Identify the earliest detectable abnormalities as likely direct effects

  • Use live imaging to capture dynamic processes in real-time

• Molecular dependency mapping:

  • Test whether CPLANE2 physically interacts with components of affected processes

  • Use proximity labeling to identify proteins in the immediate vicinity of CPLANE2

  • Determine whether interactions are direct or mediated by other complex members

• Separation-of-function mutations:

  • Design targeted mutations that affect specific domains or interactions

  • Compare phenotypes of different mutants to isolate function-specific effects

  • Use structure-guided mutations based on known or predicted protein structures

• Rapid induction systems:

  • Employ acute protein degradation systems to observe immediate consequences

  • Use optogenetic or chemical-genetic approaches for temporal control

  • Compare acute versus chronic loss-of-function phenotypes

• Biochemical reconstitution:

  • Reconstitute key steps in vitro with purified components

  • Test whether CPLANE2 is necessary and sufficient for specific biochemical activities

  • Compare activities of wild-type and variant proteins in defined biochemical assays

What are the most promising approaches for developing therapeutic interventions targeting CPLANE2-related ciliopathies?

Based on current understanding of CPLANE2 function, promising therapeutic approaches include:

• Targeted gene therapy:

  • Develop CRISPR-based approaches to correct pathogenic variants

  • Design AAV vectors suitable for affected tissues (kidney, brain, retina)

  • Consider approaches that upregulate wild-type allele expression in heterozygotes

• Small molecule screening:

  • Identify compounds that can stabilize mutant CPLANE2 proteins

  • Screen for molecules that enhance remaining GTPase activity in hypomorphic mutants

  • Develop drugs targeting downstream pathways to bypass CPLANE2 dysfunction

• Bypass strategies:

  • Identify and target downstream effectors that could circumvent CPLANE2 requirement

  • Develop approaches to modulate basal body docking through alternative pathways

  • Target compensatory mechanisms that might be activated in CPLANE2 deficiency

• Antisense oligonucleotide therapy:

  • For splicing mutations, design antisense oligonucleotides to restore proper splicing

  • Target nonsense-mediated decay to preserve partially functional truncated proteins

  • Explore exon skipping for mutations in non-essential regions

• Structure-based drug design:

  • Use structural information about CPLANE2 and the CPLANE complex to design targeted therapeutics

  • Develop molecules that can stabilize protein-protein interactions within the complex

  • Design allosteric modulators of CPLANE2 GTPase activity

What key questions remain unanswered about CPLANE2's role in ciliary biology?

Despite significant advances, several critical questions about CPLANE2 remain unanswered:

• Regulatory mechanisms:

  • How is CPLANE2 activity regulated during development and in different tissues?

  • What upstream signals control CPLANE2 function during ciliogenesis?

  • Are there tissue-specific regulatory mechanisms for CPLANE2?

• Molecular targets:

  • What are the direct effectors downstream of CPLANE2?

  • How does CPLANE2 physically connect to the basal body docking machinery?

  • What are the GTP-dependent binding partners of CPLANE2?

• Functional diversity:

  • Does CPLANE2 function differently in motile versus primary cilia?

  • Are there ciliary subtype-specific roles for CPLANE2?

  • How does CPLANE2 function change during evolution across species?

• Interaction with ciliopathy networks:

  • How does the CPLANE complex interact with other ciliopathy-associated complexes?

  • Are there genetic interactions between CPLANE2 and other ciliopathy genes?

  • Do modifiers exist that explain phenotypic variability in CPLANE2-related disorders?

• Non-ciliary functions:

  • Does CPLANE2 have functions independent of ciliogenesis?

  • Are there roles for CPLANE2 in cell cycle regulation or other cellular processes?

  • How do potential non-ciliary functions contribute to ciliopathy phenotypes?