Recombinant Xenopus laevis Cyclin-F (ccnf), partial

What is Xenopus laevis Cyclin-F and what is its role in cell cycle regulation?

Cyclin F is a substrate recognition receptor for the SCF (Skp1-Cul1-F-box protein) family of E3 ubiquitin ligases, playing a crucial role in cell cycle control through ubiquitin-dependent protein degradation . Unlike other cyclins that activate cyclin-dependent kinases (CDKs), Cyclin F primarily functions by targeting specific proteins for ubiquitination and subsequent proteasomal degradation. In Xenopus development, cyclin expression patterns are tightly regulated and exhibit tissue-specific distribution that contributes to proper embryonic development . The SCF^cyclin F complex specifically regulates proliferation by controlling the degradation of cell cycle proteins including members of the retinoblastoma (RB) protein family .

How does Cyclin F expression in Xenopus compare to other cyclins during development?

Xenopus laevis expresses multiple cyclins (including D1, D2, E, A1, A2, and B1) with distinct temporal and spatial patterns during development . Unlike other cyclins that show precise correlation with their activating CDKs, Cyclin F demonstrates unique expression patterns. While cyclins like B1 show notable oscillations during early embryonic divisions in Xenopus , Cyclin F exhibits more specialized functions in proliferation control. Additionally, maternal and zygotic cyclin forms demonstrate markedly different expression patterns throughout development . Cyclin F's role as part of the SCF complex positions it differently in the regulatory hierarchy compared to cyclins directly involved in CDK activation.

What biochemical characteristics distinguish Cyclin F from other cyclins in Xenopus?

Cyclin F contains an F-box domain that allows it to associate with the SCF complex components (Skp1, Cul1, and Roc1), forming a functional E3 ligase . Unlike cyclins that primarily function through CDK binding and activation, Cyclin F recognizes specific substrates through recognition motifs, including the Cy motif (similar to the RxL motif found in some CDK substrates) . In reconstituted systems, SCF^cyclin F works with the E2 enzymes UBCH7 and CDC34b along with the RBR E3 ligase ARIH1 to ubiquitinate targets . These distinct biochemical properties make Cyclin F a unique member of the cyclin family in Xenopus and other vertebrates.

What are the optimal methods for expressing and purifying recombinant Xenopus laevis Cyclin F?

Based on published protocols, recombinant Xenopus Cyclin F can be efficiently produced using baculovirus expression systems in Sf9 insect cells . For improved solubility, researchers often use truncated versions lacking the first ~25 amino acids and the C-terminal PEST domain (residues 25-546) . The protein can be co-expressed with Skp1 to form a stable dimer that maintains substrate recognition capability. Purification typically employs affinity chromatography with GST or His tags followed by size exclusion chromatography.

For biochemical assays, the following purification strategy has proven effective:

• Express truncated Cyclin F (25-546) with Skp1 in Sf9 cells

• Lyse cells in buffer containing protease inhibitors

• Purify using affinity chromatography

• Remove affinity tag if necessary

• Further purify by ion exchange and size exclusion chromatography

• Verify purity by SDS-PAGE and functionality through binding assays

How can researchers reconstitute functional SCF^cyclin F complexes in vitro?

To reconstitute functional SCF^cyclin F complexes for ubiquitination assays, researchers should combine the following purified components :

The reaction requires all components to achieve robust ubiquitination of substrates. As demonstrated in published studies, omission of any key component (Nedd8-Cul1-Roc1, Skp1-Cyclin F, or ARIH1/UBCH7) completely abrogates the ubiquitination activity .

What assays can be used to measure Cyclin F binding to potential substrates?

Several approaches can effectively measure Cyclin F substrate interactions:

• Fluorescence polarization anisotropy assay: This quantitative method uses fluorescently labeled peptides containing Cy motifs (e.g., TAMRA-labeled peptides) to measure direct binding to recombinant Cyclin F-Skp1 dimers. Competition assays with unlabeled potential substrates can determine binding affinities (Ki values) .

• Co-immunoprecipitation: For cellular validation, epitope-tagged Cyclin F (FLAG-Cyclin F) can be expressed in Xenopus egg extracts or cultured cells and immunoprecipitated to identify interacting proteins.

• In vitro ubiquitination assays: Reconstituted SCF^cyclin F complexes can be used to test whether potential substrates are ubiquitinated in a Cyclin F-dependent manner .

• Protein stability assays: Cycloheximide chase experiments comparing the half-life of potential substrates with or without co-expression of Cyclin F can identify proteins whose stability is regulated by Cyclin F .

How does Cyclin F coordinate with other cell cycle regulators in Xenopus embryonic development?

Cyclin F functions within a complex network of cell cycle regulators in Xenopus embryonic development. While cyclins B and E directly control CDK1 and CDK2 activation to drive mitosis and DNA replication respectively , Cyclin F operates through targeted protein degradation to coordinate these processes. In early Xenopus embryos, the cell cycle transitions from a slow first cycle to rapid subsequent divisions, with cycle length precisely regulated . Cyclin F likely contributes to this regulation by controlling the stability of key cell cycle proteins.

The Cyclin F substrate p130/RBL2 functions as a cell cycle repressor in coordination with other RB-family proteins . By targeting p130 for degradation, Cyclin F helps relieve repression of E2F-regulated genes necessary for cell cycle progression. This mechanism is particularly important for the transition between G1 and S phases, complementing the functions of other cyclins like Cyclin E, which directly activates CDK2 to trigger DNA replication .

What are the current technical challenges in studying Cyclin F function in Xenopus systems?

Several technical challenges complicate Cyclin F research in Xenopus:

• Redundancy and compensation: The presence of multiple F-box proteins and overlapping substrate specificity can mask phenotypes when Cyclin F function is disrupted.

• Temporal regulation: Cyclin F activity is likely tightly regulated during development, requiring precisely timed interventions to observe phenotypes.

• Substrate identification: Comprehensive identification of physiological substrates remains challenging, as binding studies may not reflect in vivo targeting priorities.

• Distinguishing maternal and zygotic effects: As with other cyclins, maternal and zygotic Cyclin F may have distinct expression patterns and functions , requiring careful experimental design to separate these effects.

• Integration with other regulatory systems: Understanding how Cyclin F-mediated ubiquitination coordinates with phosphorylation and other post-translational modifications requires sophisticated multi-omics approaches.

How can CRISPR/Cas9 genome editing be applied to study Cyclin F function in Xenopus laevis?

CRISPR/Cas9 genome editing offers powerful approaches to investigate Cyclin F function:

• Gene knockout studies: Despite the pseudo-tetraploid nature of X. laevis, efficient targeting of both homeologs can generate functional knockouts to assess developmental phenotypes.

• Domain-specific mutations: Precise editing can create mutations in functional domains (e.g., the F-box domain or substrate-binding regions) to distinguish different aspects of Cyclin F function.

• Fluorescent tagging: Endogenous tagging of Cyclin F enables real-time visualization of protein dynamics during development without overexpression artifacts.

• Substrate validation: Editing recognition motifs in potential substrates can confirm their dependence on Cyclin F for regulation in vivo.

When designing CRISPR experiments, researchers should consider:

• Targeting conserved regions to affect both homeologs simultaneously

• Using multiple guide RNAs to increase editing efficiency

• Including appropriate controls to account for off-target effects

• Validating edits through sequencing and functional assays

What insights can Xenopus Cyclin F studies provide for understanding human diseases?

Studies of Xenopus Cyclin F have significant implications for understanding human diseases, particularly cancer and developmental disorders. The SCF^cyclin F complex regulates the retinoblastoma protein family, which are key tumor suppressors . Dysregulation of this pathway is implicated in multiple cancer types. Additionally, as developmental timing and cell proliferation are tightly regulated processes, abnormalities in Cyclin F function could contribute to developmental disorders.

The advantages of using Xenopus as a model system include:

• The ability to manipulate embryonic development through microinjection

• The capacity to perform biochemical studies in egg extracts

• Evolutionary conservation of cell cycle machinery between Xenopus and humans

• The ability to examine tissue-specific effects in a developing vertebrate

How do mutations in Cyclin F recognition motifs affect substrate binding and cell cycle progression?

Mutations in Cyclin F recognition motifs significantly impact substrate binding and downstream cell cycle effects. For example, mutation of the RxL motif in p130 (specifically R680 and L682 to alanines) reduced binding affinity to Cyclin F by approximately 100-fold (Ki = 28 ± 5 μM for the mutant versus 0.44 ± 0.04 μM for wild-type) . These mutations prevented SCF^cyclin F-mediated ubiquitination of p130 in vitro and dramatically increased protein stability in vivo .

The functional consequences of preventing Cyclin F-mediated degradation include:

• Enhanced repression of cell cycle gene expression

• Reduced cellular proliferation

• Altered timing of cell cycle transitions

• Potential developmental abnormalities due to improper cell cycle control

What approaches can be used to map the complete interactome of Xenopus Cyclin F during development?

Mapping the complete Cyclin F interactome requires integrating multiple complementary approaches:

• Proximity-based labeling: BioID or TurboID fusions with Cyclin F can identify proteins in close proximity in living cells or embryos.

• Quantitative proteomics: Stable isotope labeling combined with immunoprecipitation can identify proteins whose abundance changes upon Cyclin F depletion or overexpression.

• Ubiquitome analysis: Global ubiquitination profiling using diGly remnant antibodies can identify substrates whose ubiquitination depends on Cyclin F.

• Developmental stage-specific analysis: Performing these studies across different developmental stages can reveal stage-specific Cyclin F substrates and interactions.

• Computational prediction: Bioinformatic screening for proteins containing conserved Cy motifs can identify candidate substrates for validation.

• Spatial resolution: Tissue-specific analysis can identify cell type-specific Cyclin F interactions relevant to developmental processes.

How do the functions of Xenopus Cyclin F compare with those in other model organisms?

Cyclin F functions show both conservation and divergence across species:

The conservation of the SCF^cyclin F complex targeting RB-family proteins suggests this is an evolutionarily ancient mechanism for controlling cell proliferation , while species-specific targets likely reflect adaptations to different developmental strategies.

What experimental approaches can distinguish between maternal and zygotic roles of Cyclin F in Xenopus development?

Distinguishing maternal and zygotic Cyclin F contributions requires specialized approaches:

• Morpholino knockdown: Targeting Cyclin F mRNA with antisense morpholinos can deplete maternal transcripts without affecting the zygotic genome .

• CRISPR/Cas9 F0 screens: Injecting Cas9 protein with guide RNAs targeting Cyclin F into fertilized eggs can disrupt the zygotic gene while leaving maternal products intact.

• Maternal-effect screens: Generating heterozygous females through gene editing and examining the phenotypes of their offspring can reveal maternal contributions.

• Rescue experiments: Selective rescue of either maternal or zygotic function through timed expression of wild-type or mutant Cyclin F can determine when and where each pool is required.

• Transcript analysis: RT-PCR with primers specific to maternal or zygotic transcripts (utilizing UTR differences) can track the transition between these pools.

How does post-translational regulation of Cyclin F differ between Xenopus and mammalian systems?

Post-translational regulation of Cyclin F shows both conserved and divergent mechanisms between Xenopus and mammals:

• Phosphorylation: In both systems, CDK-mediated phosphorylation likely regulates Cyclin F stability and activity, though specific sites may differ.

• Ubiquitination: Self-ubiquitination as an auto-regulatory mechanism appears conserved, but the E2 enzymes involved may vary between species.

• Subcellular localization: Nuclear localization signals are conserved, but regulation of nuclear-cytoplasmic shuttling may be adapted to the rapid cell cycles in early Xenopus embryos compared to mammalian somatic cells.

• Protein-protein interactions: While core SCF components (Skp1, Cul1) interactions are conserved, accessory factors may differ between species.

• Stability control: The rapid cell cycles in early Xenopus development may necessitate distinct regulation of Cyclin F protein turnover compared to the slower cycles in mammalian systems.