Deciphering phase separation mechanisms during multiple centrioles production
Multiciliated cells (MCCs) are widespread in evolution and serve a large spectrum of physiological functions ranging from locomotion in marine larva to mucus transport in human airways. A key step in MCC development is the production of multiple centrioles, the structure at the base of a cilum, that template dozens to hundreds of cilia at the apical surface. Seminal electron microscopy studies have reported that this process is characterized by the successive formation of two atypical membrane-less organelles: fibrous granules (FGs) and deuterosomes. Recent work suggests that fibrous granules are biomolecular condensates that form via Liquid-Liquid Phase Separation (LLPS).
The goal of this project is to decipher the biological and physical mechanisms that drive the Liquid- Liquid phase separation to understand how MCC regulates to the formationof FGs and control the number of produced centrioles. In this project, we will combine cell biology and soft matter physics to decipher the biophysical mechanisms involved in multiple centriole production.
Centrioles – fibrous granules – deuterosomes - liquid-liquid phase separation – minimal biomimetic system
Our objective is to characterize the regulatory mechanisms of the phase transition in MCCs to understand their impact on the control of centriole number. Based on preliminary results, we postulate that nuclear folds and the kinases PLK1 and Dyrk3 modulate FG condensation. To test this, we defined three objectives: i) quantify the relationship between the nucleus and FGs in MCC subtypes producing different numbers of centrioles; ii) quantify the dynamics of fibrous granule formation/dissolution and their modulation by PLK1 and Dyrk3 kinases in vivo; iii) determine the minimal biophysical mechanisms involved in the formation/dissolution of FGs by using a minimal system reconstituted in vitro.
Proposed approach (experimental / theoretical / computational)
We will combine cell biology and soft matter physics to decipher the biophysical mechanisms involved in multiple centriole production. Probing LLPS in vivo (team 1): The PhD student will use live-confocal and advance image analysis to characterize the relationship between nucleus and FG in subtypes of MCC producing different numbers of centrioles. Probing LLPS in vitro (team 2): Starting from a limited set of purified biochemical components identified by team 1 we will 1) reconstruct, in cell-sized droplets, the LLPS observed in-vivo, 2) quantify the physical properties (protein diffusion, saturation concentration, viscosity, surface tension) of the in vitro liquid organelles using advanced microscopy (FRAP, FCS) and micro-rheology. 3) by varying the concentration of encapsulated components we will establish the phase diagram of LLPS. We will identify the different regimes of formation/dissolution of FG in-vitro and decipher the driving force underlying such regimes.
Results obtained thought these approaches will feed a model, developed in collaboration with a theoretician (Pierre Ronceray, CINaM) aiming at establishing the general laws governing in vivo LLPS associated with multiple centriole production.
We propose a collaboration between two Centuri groups with long standing interest in multiciliated epithelia. The biologists at IBDM (team 1) have developed multiple research projects aiming at understanding the molecular mechanisms of centriole production and organization in multiciliated cells. Experimental approaches mastered by this team include advanced cell biology and proteomic analyses, in vivo functional manipulation in mouse and Xenopus animal models, advanced imaging techniques and quantitative biology approaches. The Physicists at CINAM (team 2) are specialists of the self-organization and coordination of cilia to generate directed flow at long distance in the bronchial epithelium. They hold a great expertise in the design of minimal systems to reconstruct and identify the minimal physical parameters involved in a biological function. The convergence of research themes of the two partners as well as the complementarity of their expertise generate a strong consortium to address LLPS in the context of multiple centriole production.
We are seeking a candidate with strong background either in Cell and Developmental Biology or in experimental physics (e.g. soft matter, biophysics…). She/he would eventually have some basic experimental skills in cell culture, microscopy and image processing., A keen interest in integrative quantitative biology and interdisciplinary research is expected. Through this project, the candidate will interact with researchers from both communities and will therefore acquire useful skills to apprehend the experimental reality in biology and physics.
Is this project the continuation of an existing project or an entirely new one? In the case of an existing project, please explain the links between the two projects
This is a new project.
2 to 5 references related to the project
- Boutin C, Kodjabachian L. Biology of multiciliated cells. Curr Opin Genet Dev. 2019 Jun;56:1-7. doi: 10.1016/j.gde.2019.04.006. Epub 2019 May 16.
- Boeynaems, S., et al., Protein Phase Separation: A New Phase in Cell Biology. Trends Cell Biol, 2018. 28(6): p. 420-435.
- Beutel, O., et al., Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions. Cell, 2019. 179(4): p. 923-936 e11.
- AA Hyman et al., Liquid-liquid phase separation in Biology, Annual review of cell and developmental biology, 2014, https://doi.org/10.1146/annurev-cellbio-100913-013325
3 main publications from each PI over the last 5 years
Biology of multiciliated cells
- Boutin C, Kodjabachian L. Curr Opin Genet Dev. 2019 Jun;56:1-7. doi: 10.1016/j.gde.2019.04.006. Epub 2019 May 16.PMID: 31102978
CDC20B is required for deuterosome-mediated centriole production in multiciliated cells.
- Revinski DR*, Zaragosi LE*, Boutin C*, Ruiz-Garcia S, Deprez M, Thomé V, Rosnet O, Gay AS, Mercey O, Paquet A, Pons N, Ponzio G, Marcet B, Kodjabachian L, Barbry P.Nat Commun. 2018 Nov 7;9(1):4668. doi: 10.1038/s41467-018-06768-z.
A dual role for planar cell polarity genes in ciliated cells.
- Boutin C, Labedan P, Dimidschstein J, Richard F, Cremer H, André P, Yang Y, Montcouquiol M, Goffinet AM, Tissir F.Proc Natl Acad Sci U S A. 2014 Jul 29;111(30):E3129-38. doi:10.1073/pnas.1404988111. Epub 2014 Jul 14.
Active mucus-cilia hydrodynamic coupling drives self-organisation of human bronchial epithelium
- E Loiseau et al., Nat. Phys., 2020 doi: 10.1038/s41567-020-0980-z
Shape Remodelling and Blebbing of Active Cytoskeletal Vesicles
- E Loiseau et al., Science Advances. (2016) doi : 10.1126/sciadv.1500465
Topology and dynamics of active nematic vesicles
- F Keber*, E Loiseau*, T Sanchez* et al., Science (2014) doi:10.1126/science.1254784