Functional proteomics of multiciliated cells – MCCproteome

The MCCproteome project aims at understanding how multiciliated cells (MCCs) are built, a question of high relevance to human health. MCCs harbor myriads of motile cilia beating coordinately to generate directional flows of biological fluids, or to move particles and cells, at the surface of specialized epithelial tissues. In human, MCCs are involved in circulation of the cerebro-spinal fluid in the central nervous system, in transportation of gametes, and in evacuation of soiled mucus from upper airways. Mutations in genes necessary for multiple cilia formation have been shown to be responsible for familial syndromes characterized by chronic airway infections, and an elevated risk of infertility. The severity of these symptoms points to the importance of studying fundamental principles of MCC biology.

In recent years, the global MCC transcriptome has been decrypted in Xenopus, mouse and human. It is now time to elucidate the functional MCC proteome, which is the main objective of this project.

Ciliogenesis is initiated when a centriole migrates underneath the cell membrane, is anchored to an actin-based scaffold and matures into a basal body, which acts as a microtubule-organizing structure necessary for axoneme extension. In the case of multiciliogenesis, a key additional step consists of a massive increase in the number of centrioles after permanent exit from the cell cycle. Our project is designed to shed light on this step, which is unique to MCCs and still poorly understood.

In vertebrate MCCs, bulk centriole biogenesis occurs around poorly characterized platforms, called deuterosomes, although modest multiplication from parental centrioles also occurs. Deuterosomes have been described five decades ago by electron microscopy as non-centriolar globular structures. A first core deuterosome component, called Deup1 was identified in 2013. Recent work in Kodjabachian’s team has identified Pericentrin and ?-Tubulin as additional deuterosome components. These studies have opened the way to further molecular and functional characterization of deuterosomes, which is the overarching objective of our project.

We will use three experimental paradigms to study deuterosome biology. i. The Xenopus embryonic epidermis, which is covered with functional MCCs 24h after fertilization, and represents a powerful and simple model to study multiciliogenesis in vivo. ii. A newly established Xenopus inducible cell line, which provides homogeneous and synchronized populations of MCCs. iii. Mouse post-natal ependymal MCCs, well-suited for in vivo fluorescent imaging and amenable to functional manipulation by electroporation. We will use a combination of super-resolution fluorescent microscopy, 2D and 3D electron microscopy, proteomics and functional manipulations to study deuterosome molecular composition, architecture, and activity.

To date, very few studies have addressed deuterosome biology at the molecular level. Through this project, our consortium is poised to make important contributions in this field. Regarding its relevance to human health, this project may help to identify potential candidate genes for diseases caused by reduced multiciliogenesis.