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bioRxiv · 10.1101/2024.04.02.587733

Interaction between ciliary component proteins from Chlamydomonas revealed by CRISPR/CAS9, cryo-electron tomography and mass spectrometry

Luo, L.·Zimmermann, N.·Noga, A.·Leitner, A.·Ishikawa, T.
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Abstract

To understand molecular mechanism of ciliary beating motion, knowledge of location, interaction and dynamics of >400 component proteins are indispensable. While recent progress of structural biology revealed conformation and localization of >100 proteins, we still need to investigate their networking, art of their interaction and assembly mechanism. We applied CRISPR/CAS9 genome editing technique to the green algae Chlamydomonas to engineer a deletion mutant of a ciliary component, FAP263, located at the distal protrusion, and examined it structurally by cryo-electron tomography (cryo-ET) and mass spectrometry (MS). Cryo-ET and atomic model fitting demonstrated that the FAP263 deletion mutant lacks additional components, FAP78, and FAP184. Unassigned density near FAP263 in the cryo-ET map of WT cilia is likely FAP151, as suggested by cross-linking mass spectrometry. Based on the structure, we modeled how these four proteins might form a complex. Furthermore, it was shown that dynein f phosphorylation is inhibited in the FAP263 mutant, indicating an important role of this protein complex for dynein f phosphorylation. Our study demonstrates a novel approach to investigate protein networking inside cilia.

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Luo, L., Zimmermann, N., Noga, A., Leitner, A., Ishikawa, T.. 2024-04-03. Interaction between ciliary component proteins from Chlamydomonas revealed by CRISPR/CAS9, cryo-electron tomography and mass spectrometry. https://doi.org/10.1101/2024.04.02.587733

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