Myristoylated Neuronal Calcium Sensor-1 captures the preciliary vesicle at distal appendages

Area of Science:

• Cell Biology
• Molecular Biology
• Organelle Biogenesis

Background:

• Primary cilia are essential microtubule-based organelles involved in various cellular functions.
• Cilium formation requires precise recruitment of preciliary vesicles to the mother centriole's distal appendage.
• The exact mechanism of preciliary vesicle capture by the distal appendage remained elusive.

Purpose of the Study:

• To elucidate the molecular mechanism by which the distal appendage recruits preciliary vesicles.
• To identify binding partners of CEP89 involved in preciliary vesicle recruitment.
• To understand the role of Neuronal Calcium Sensor-1 (NCS1) in primary cilium assembly.

Main Methods:

• Co-immunoprecipitation to identify CEP89 interactors.
• Immunofluorescence microscopy to determine protein localization.
• CRISPR/Cas9 gene editing to create knockout cell lines (CEP89 and NCS1).
• Analysis of cilium formation and vesicle recruitment in knockout cells and cells expressing mutant NCS1.

Main Results:

• Neuronal Calcium Sensor-1 (NCS1) was identified as a stoichiometric interactor of CEP89.
• CEP89 recruits NCS1 to the distal appendage, bridging the gap to the vesicle marker RAB34.
• NCS1 is crucial for preciliary vesicle recruitment and subsequent cilium formation, dependent on its myristoylation motif.

Conclusions:

• The study reveals the first identified mechanism for distal appendage-mediated preciliary vesicle recruitment.
• CEP89 and NCS1 form a critical complex for initiating primary cilium assembly.
• NCS1's myristoylation is essential for its function in recruiting vesicles to the distal appendage.