Self-organization of the cilium: the role of the tubulin code

Project Information

CiliaTubulinCode

Grant agreement ID: 819826

DOI

10.3030/819826

Project closed

EC signature date 6 February 2019

Start date 1 March 2019

End date 31 October 2024

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 986 406,00

EU contribution

€ 1 986 406,00

Coordinated by

FONDAZIONE HUMAN TECHNOPOLE
Italy

Periodic Reporting for period 5 - CiliaTubulinCode (Self-organization of the cilium: the role of the tubulin code)

Reporting period: 2024-02-01 to 2024-10-31

Summary of the context and overall objectives of the project

CiliaTubulinCode: Deciphering the Role of the Tubulin Code in Cilia Biology.
The CiliaTubulinCode project investigates how tubulin post-translational modifications (PTMs) contribute to the self-organization, mechanics, and function of motile cilia—specialized organelles crucial for cellular motility and signaling. Cilia play an essential role in many physiological processes across eukaryotic organisms, including humans, where ciliary dysfunction leads to severe diseases such as primary ciliary dyskinesia, infertility, and neurodevelopmental disorders.
Understanding the role of tubulin PTMs in cilia presents significant challenges due to the complexity and nanoscale organization of the ciliary axoneme. To overcome these difficulties, CiliaTubulinCode integrates a cutting-edge combination of approaches, including:
- Advanced imaging techniques, such as correlative light and electron microscopy (CLEM), immuno-cryo-electron tomography (cryo-ET), and ultrastructural expansion microscopy.
- Molecular and biochemical methods, including CRISPR-Cas genome editing, quantitative mass spectrometry, and in vitro reconstitution of ciliary processes.
- Live-cell imaging and single-molecule tracking, enabling the study of dynamic ciliary processes at high spatiotemporal resolution.

Objectives of CiliaTubulinCode.
The project focuses on three major objectives:
1. Understanding the role of the tubulin code in the regulation of intraflagellar transport (IFT)—the bidirectional transport system essential for cilia assembly and maintenance.
2. Investigating how the tubulin code contributes to axoneme assembly and the regulation of axonemal components, such as dyneins, which generate the forces required for ciliary beating.
3. Creating a high-resolution spatiotemporal map of tubulin PTMs in motile cilia and eukaryotic flagella, shedding light on their role in structural integrity and function.
The results of this research are of broad interest, extending beyond cilia and microtubule biology to the larger fields of cell biology, molecular biology, and biophysics. Moreover, this work provides fundamental insights into human diseases linked to mutations in tubulin PTM enzymes, reinforcing its clinical relevance.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Key findings and contributions of this project are:
i) The development of genetic tools in Chlamydomonas. We established a CRISPR-Cas system for efficient gene knock-ins/knock-outs that enabled precise tagging and functional analysis of PTM enzymes, IFT proteins, and axonemal components.
ii) The visualisation of the Ciliary Tubulin Code at high resolution. We developed a cryo-immunolabeling electron microscopy method to map tubulin PTMs at sub-microtubular resolution. With this method we discovered protofilament-specific patterns of tubulin PTMs, and revealed their role in axonemal organization and ciliary motility. In particular we identified the role of glycylation in dynein regulation and polyglutamylation in structural integrity through the interaction with the nexin-dynein regulatory complex.
iii) The structural role of tubulin PTMs. Contrary to initial hypotheses, we found that PTMs did not determine axonemal protein positioning but were instead influenced by axonemal components.
iv) The Impact of the Tubulin Code on Intraflagellar transport (IFT). By knocking out the enzyme responsible of tubulin detyrosination and a new method to reactivate entire IFT trains in vitro, we demonstrated that PTMs regulate the spatial segregation of IFT trains. Disrupted PTM patterns led to train collisions and defective ciliary assembly in cells. In vitro assays confirmed PTM-dependent motor selection for IFT transport.
v) Mechanisms of tubulin PTM patterning. Ongoing research in the lab focuses on how PTMs patterns are established, including the role of IFT and microtubule internal proteins in modifying enzyme localization and activity.
This project has significantly advanced our understanding of the tubulin code in motile cilia, revealing its impact on structural organization, transport regulation, and ciliary function.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Impact and Future Directions
The CiliaTubulinCode project has significantly advanced our understanding of the tubulin code in motile cilia, with profound implications for structural organization, transport regulation, and ciliary mechanics. These findings have expanded our knowledge of microtubule biology and are likely to influence research on other cytoskeletal systems.

Moving forward, our research will focus on:
1. Deciphering how PTMs influence molecular motors beyond IFT, including dynein and kinesin dynamics.
2. Exploring PTM-dependent regulation in other cytoskeletal structures, such as mitotic spindles and neuronal microtubules.
3. Investigating potential therapeutic strategies for cilia-related disorders, based on our mechanistic insights into PTM function.
By integrating molecular genetics, high-resolution imaging, and biochemical reconstitution, CiliaTubulinCode has provided a detailed framework for understanding how tubulin modifications shape ciliary function and contribute to human health and disease.

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