Recombinant Danio rerio Centrosomal protein of 41 kDa (cep41)

What is CEP41 and what is its primary function in zebrafish?

CEP41 (Centrosomal protein of 41 kDa) is a protein predominantly localized to centrioles and cilia in zebrafish. It plays a critical role in ciliary tubulin glutamylation, which is essential for maintaining proper ciliary structure and motility. In zebrafish, cep41 is expressed in various ciliary organs including Kupffer's vesicle (KV), ear, heart, brain, and kidney - regions predominantly affected in ciliopathies such as Joubert syndrome .

CEP41 functions as a microtubule-associated protein with microtubule-stabilizing activity. It binds to preformed microtubules, promotes microtubule nucleation, and suppresses microtubule disassembly . This function is essential for maintaining the integrity of ciliary axonemes and proper cilia-dependent signaling.

What is the domain structure of zebrafish CEP41 and how does it relate to function?

Zebrafish CEP41 contains several functional domains that are highly conserved across species:

• N-terminal region with coiled-coil motifs: Essential for CEP41's interaction with microtubules

• Rhodanese homology domain (RHOD): Critical for CEP41-microtubule interaction; catalytically inactive but contributes to protein stability and interactions

• C-terminal disordered region: Appears dispensable for CEP41-microtubule interaction

The functional significance of these domains has been demonstrated through deletion studies:

• Deletion of the N-terminal region (ΔN) results in diffuse cytoplasmic localization rather than microtubule association

• Deletion of the RHOD domain (ΔRHOD) leads to substantial protein aggregation

• Deletion of the C-terminal region still allows for protein localization to microtubules

These structural features explain how CEP41 interacts with microtubules to facilitate tubulin glutamylation specifically in cilia.

What are effective methods for knocking down or knocking out CEP41 in zebrafish?

Several approaches have been validated for CEP41 depletion in zebrafish:

1. Morpholino antisense oligonucleotides (MOs):

• Translation-blocking MOs targeting the start site: 5′-CATCTTCCAGCAGCAGAGCTTCGGC-3′

• Efficiency verification: Western blot assay comparing CEP41 protein levels in control and morphant zebrafish

• Typical concentration: 2.5 µg/µl, microinjected using a pneumatic microinjection system

2. Splice-blocking MOs:

• Target splice junctions in cep41 pre-mRNA, leading to aberrant splicing and non-functional protein

• Validation: RT-PCR to confirm altered splicing patterns (detailed in Appendix Fig S2A–E)

3. CRISPR/Cas9 gene editing:

• Successfully used to generate cep41-knockout zebrafish lines

• Implementation steps detailed in Appendix Fig S2F–I

• Validation through sequencing and phenotypic analysis

Researchers should consider experimental timeframe, specificity requirements, and potential off-target effects when selecting a method.

How can I perform rescue experiments in CEP41-deficient zebrafish?

Rescue experiments provide critical evidence for specificity of observed phenotypes and allow functional assessment of specific mutations:

Protocol:

• Synthesize capped mRNAs of wild-type or mutated human CEP41 variants using mMESSAGE mMACHINE kit (Ambion)

• Co-inject synthesized mRNAs (150–300 ng/µl) together with cep41 MOs into one-cell stage zebrafish embryos

• Assess phenotypic rescue through appropriate assays:

  • For vascular phenotypes: Examine vessel formation in Tg(kdrl:eGFP) embryos

  • For ciliary function: Analyze cilia structure, tubulin glutamylation, and motility

  • For body morphology: Document curved body axis, hydrocephalus, and other morphological features

This approach allows testing whether human CEP41 can functionally substitute for zebrafish CEP41 and examining the consequences of specific mutations.

Note: Representative data based on synthesized information from multiple studies

How does CEP41 specifically regulate tubulin glutamylation in cilia but not in cytoplasm?

CEP41 plays a specialized role in regulating tubulin glutamylation specifically in the ciliary compartment through several mechanisms:

• Subcellular localization: CEP41 predominantly localizes to centrioles and cilia, positioning it to influence ciliary microtubule modifications specifically

• Compartmentalized function: The ciliary compartment is biochemically distinct from the cytoplasm, allowing for differential regulation of tubulin modifications

• Substrate specificity: CEP41 may interact with or influence the activity of tubulin glutamylases (TTLLs) specifically in the ciliary environment, potentially through direct binding or scaffold functions

• Experimental evidence:

  • CEP41-deficient zebrafish and human cells show reduced glutamylation of ciliary tubulin without affecting cytoplasmic tubulin glutamylation

  • Other ciliopathy-related proteins (like INPP5E) displayed no such glutamylation defect, suggesting specificity to CEP41 function

  • The hypoglutamylation caused by CEP41 deficiency can be rescued by depletion of the deglutamylase CCP5

This specialized function explains why CEP41 mutations primarily affect ciliated tissues and result in ciliopathy phenotypes.

What is the relationship between CEP41, tubulin glutamylation, and ciliary structure in zebrafish?

Research has established clear connections between CEP41 function, tubulin glutamylation levels, and ciliary structural integrity:

• Structural abnormalities: Transmission electron microscopy (TEM) of CEP41-deficient zebrafish reveals specific structural defects in ciliary axonemes:

  • Collapsed and/or duplicated A tubules of the outer doublet microtubules

  • These defects are absent in control cilia with normal glutamylation levels

• Functional consequences: The structural abnormalities directly impact ciliary function:

  • Disabled motility of both Kupffer's vesicle (KV) and kidney cilia in cep41 morphants

  • Defective mechanosensation in endothelial cilia

• Rescue evidence: Depletion of the deglutamylase CCP5 in CEP41-deficient zebrafish restores:

  • Tubulin glutamylation levels

  • Vascular development

  • Other ciliopathy-related phenotypes

This relationship demonstrates how molecular-level defects in tubulin modification translate to tissue and organ-level developmental abnormalities.

What is the role of CEP41 in zebrafish vascular development, and how can it be studied?

CEP41 plays a critical role in zebrafish vascular development through regulation of endothelial cell function:

Key vascular phenotypes in CEP41-deficient zebrafish:

• Thin, short, fused, or missing intersegmental vessels (ISVs)

• Ruptured dorsal longitudinal anastomotic vessels (DLAVs)

• Defective caudal vein plexus (CVP) formation with reduced venous sprouts and vascular loops

Experimental approaches to study CEP41's role in vascular development:

• Transgenic reporter lines:

  • Tg(kdrl:eGFP) zebrafish with fluorescently labeled vascular endothelial cells allow direct visualization of vessel formation

  • Quantification of vessel parameters (length, branching, connections) at specific developmental timepoints (e.g., 40 hpf for ISVs/DLAVs)

• Live imaging:

  • Time-lapse microscopy to track vessel sprouting, extension, and anastomosis in real-time

  • Analysis of endothelial cell migration patterns and dynamics

• Molecular analysis:

  • qRT-PCR for angiogenic factors (e.g., vegfa, vegfr2) at different developmental stages

  • Immunostaining for signaling pathway components (e.g., phosphorylated AURKA)

• Rescue experiments:

  • Co-injection of cep41 MOs with mRNAs encoding wild-type or mutant CEP41

  • Depletion of CCP5 (deglutamylase) to restore tubulin glutamylation and assess vascular rescue

• Functional vascular assessments:

  • Blood flow dynamics using transgenic lines with labeled blood cells

  • Vascular permeability assays

These approaches have revealed that CEP41 regulates vascular development through its effects on endothelial cilia function and mechanotransduction.

How does CEP41 mediate mechanosensation in endothelial cells and influence angiogenesis?

CEP41 plays a crucial role in the endothelial response to fluid shear stress, linking mechanical stimuli to angiogenic responses:

Mechanistic pathway:

• Ciliary mechanosensation:

  • Endothelial cilia sense blood flow-induced shear stress

  • CEP41 maintains proper ciliary tubulin glutamylation, essential for mechanosensory function

  • CEP41-deficient cells fail to respond properly to shear stress, retaining cobblestone appearance instead of elongating and aligning with flow direction

• Signal transduction:

  • Properly functioning cilia activate Aurora Kinase A (AURKA) in response to shear stress

  • CEP41-depleted cells and zebrafish show reduced AURKA activation when exposed to shear stress

  • AURKA activation is necessary for appropriate angiogenic responses

• Transcriptional regulation:

  • Mechanosensation through CEP41-dependent cilia influences expression of pro-angiogenic genes

  • CEP41-deficient zebrafish show dampened upregulation of vegfa and vegfr2 mRNAs when exposed to shear stress

  • This reduced expression correlates with defective vessel formation

• Cellular responses:

  • Control endothelial cells enhance migration and tube formation under shear stress

  • CEP41-depleted cells show no such enhancement, failing to respond to mechanical cues

  • These cellular defects directly contribute to vascular malformations

Experimental evidence connecting mechanosensation to angiogenesis:

• Shear stress experiments using a verified fluid flow model (≥15 dynes/cm²) demonstrate CEP41's role in mechanotransduction[5,

• Timing experiments show that CEP41 deficiency has minimal effects at 18 hpf (minimal shear stress) but significant effects at 26 hpf (>0.5 dynes/cm²)

• Rescue of CEP41-depleted phenotypes with constitutively active AURKA confirms the mechanistic pathway

This mechanistic understanding connects CEP41's molecular function to physiological angiogenesis and provides insights into ciliopathy vascular phenotypes.

How can zebrafish CEP41 models contribute to understanding human Joubert syndrome?

Zebrafish CEP41 models provide valuable insights into Joubert syndrome pathogenesis and potential therapeutic approaches:

Phenotypic parallels:

• CEP41-deficient zebrafish display multiple features that parallel Joubert syndrome, including:

  • Brain abnormalities

  • Curved body axis

  • Left-right asymmetry defects (e.g., heart looping abnormalities)

  • Defective ciliary structure and function

Mutation analysis:

• Several disease-causing mutations identified in Joubert syndrome patients have been studied in zebrafish:

  • R179H mutation in the RHOD domain abolishes microtubule localization

  • Other mutations (M36T, Q89E, P206A) maintain microtubule localization despite causing disease

• These functional differences may explain phenotypic variability in human patients

Mechanistic insights:

• Zebrafish studies have revealed the molecular basis of CEP41-associated disease:

  • Defective ciliary tubulin glutamylation

  • Structural abnormalities in ciliary axonemes

  • Impaired ciliary motility and function

  • Disrupted cell cycle progression

Therapeutic implications:

• Depletion of the deglutamylase CCP5 rescues phenotypes in CEP41-deficient zebrafish

• This suggests potential therapeutic approaches focused on restoring balanced tubulin glutamylation

• The zebrafish model enables screening of compounds that might rescue CEP41-associated defects

Modifier discovery:

• Joubert syndrome patients with homozygous CEP41 mutations display the characteristic "molar tooth sign"

• Heterozygous CEP41 mutations have been found in patients with other ciliopathies like Bardet-Biedl syndrome (BBS)

• This suggests CEP41 may function as a genetic modifier across ciliopathy spectrum

By combining genetic, cellular, and organismal approaches, zebrafish CEP41 models provide a comprehensive platform for studying Joubert syndrome pathogenesis.

How do mutations in different domains of CEP41 differentially affect protein function?

Different mutations in CEP41 have distinct effects on protein function, explaining phenotypic variability in ciliopathy patients:

1. Rhodanese homology domain (RHOD) mutations:

The R179 residue appears to be particularly critical, forming multiple polar contacts with neighboring residues that maintain structural integrity necessary for microtubule binding .

2. N-terminal coiled-coil region mutations:

Despite maintaining microtubule association, these mutations likely disrupt other aspects of CEP41 function, possibly including protein-protein interactions .

3. Splice site mutations:

Homozygous splice site mutations identified in Joubert syndrome patients:

• c.97+3_5delGAG: Abolishes splice donor site from exon 2, leading to exon skipping and premature stop codon

• c.423-2A>C: Abolishes splice acceptor site from exon 7

These mutations likely result in complete loss of functional protein.

Functional consequences in zebrafish:

• Complete loss of CEP41 results in severe ciliopathy phenotypes

• Expression of human CEP41 with specific mutations in zebrafish can reveal domain-specific effects on:

  • Ciliary tubulin glutamylation

  • Axonemal structure

  • Ciliary motility

  • Left-right asymmetry

  • Vascular development

Understanding these structure-function relationships can help predict disease severity in patients and guide potential therapeutic approaches.

What is the role of CEP41 in cell cycle regulation and how does this impact development?

Beyond its ciliary functions, CEP41 plays critical roles in cell cycle regulation that impact development:

Experimental findings:

• Cell proliferation:

  • CEP41 depletion inhibits cell proliferation by 72 ± 17% compared to 20 ± 9% in control cells

  • This dramatic reduction in proliferation has significant implications for developmental processes

• Cell cycle distribution:

  • CEP41-depleted cells show significantly reduced G2/M phase population (21 ± 2% vs. 29 ± 2% in controls)

  • Slight increase in S phase cells suggests potential S-G2 transition defects

• Cell cycle progression:

  • When synchronized at G1/S boundary and released:

    • Control cells: 45 ± 1% progress to G2/M after 8 hours

    • CEP41-depleted cells: only 27 ± 2% reach G2/M after 8 hours

  • 54.3 ± 0.5% of CEP41-depleted cells remain in G1 post-release (vs. 40 ± 2% of controls)

Molecular mechanisms:

• CEP41's microtubule-stabilizing activity is essential for proper mitotic spindle formation

• Its centrosomal localization positions it to influence spindle pole organization

• CEP41 may regulate microtubule dynamics during cell division

• Cell morphology changes in CEP41-depleted cells (55 ± 7% reduction in cytoplasmic area) may impair mitotic processes

Developmental impacts:

• Reduced proliferation rates in developing tissues can lead to:

  • Smaller organ size

  • Delayed developmental timing

  • Altered tissue architecture

• These effects may contribute to the developmental abnormalities seen in ciliopathies

• May partially explain why CEP41 mutations affect multiple organ systems during embryogenesis

This cell cycle regulatory function represents an important aspect of CEP41 biology that extends beyond its ciliary roles and has significant implications for developmental processes.

How can the interaction between CEP41 and tubulin deglutamylases be exploited for potential therapeutic approaches?

The functional antagonism between CEP41 and tubulin deglutamylases (particularly CCP5) presents promising therapeutic opportunities:

Molecular relationship:

• CEP41 promotes ciliary tubulin glutamylation

• CCP5 (Cytosolic Carboxypeptidase 5) removes glutamate residues from tubulin

• CEP41 deficiency leads to hypoglutamylation of ciliary tubulin

• CCP5 depletion increases tubulin glutamylation

Experimental evidence for therapeutic potential:

• In vitro rescue:

  • HUVECs co-transfected with CEP41-specific and CCP5-specific siRNAs show:

    • Rescue of defective cell migration

    • Restoration of tubulogenesis

    • Recovery of normal responses to mechanical stimuli

• In vivo rescue:

  • cep41-knockdown/knockout zebrafish injected with ccp5 MOs show:

    • Rescue of impaired ISVs and DLAVs formation

    • Restoration of vascular network development

  • This proves the principle that modulating deglutamylase activity can compensate for CEP41 dysfunction

• Molecular verification:

  • CCP5 silencing enhances ciliary tubulin glutamylation

  • Restores reduced tubulin glutamylation in CEP41-depleted cilia

  • Does not affect ciliation rate (ARL13b-labeled cells)

Therapeutic avenues:

• Small molecule inhibitors:

  • Development of specific CCP5 inhibitors could restore balanced tubulin glutamylation

  • High-throughput screening using zebrafish CEP41 models could identify effective compounds

• Genetic approaches:

  • Targeted reduction of CCP5 expression through antisense oligonucleotides or RNA interference

  • CRISPR-based approaches to modify CCP5 activity

• Combination strategies:

  • Partial inhibition of multiple deglutamylases (CCP1-6)

  • Simultaneous enhancement of glutamylases (TTLLs)

• Delivery challenges:

  • Targeting specific tissues affected in ciliopathies

  • Achieving appropriate developmental timing of intervention

  • Avoiding off-target effects on cytoplasmic tubulin glutamylation

This therapeutic approach based on tubulin modification balance represents a promising direction for treating CEP41-related ciliopathies and potentially other ciliopathies with similar molecular defects.