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Extracellular vesicle production maintains ciliary protein homeostasis

par Razzauti Sanfeliu, Adrià
Président du jury Remmelink, Myriam
Promoteur Laurent, Patrick
Publication Non publié, 2023-04-06

Thèse de doctorat

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Résumé :

In the nervous system, glial cells coexist and collaborate with neurons to process information and generate adaptive responses. However, how neurons and glia in the nervous system communicate with each other to properly perform their functions is an underexplored research topic. The survival of neurons depends on glia making it difficult to analyze intercellular communication. Here, I used the nematode Caenorhabditis elegans to circumvent these obstacles and study neuron-glial communication. Taking advantage of the invariant anatomy of C. elegans, I selected as a model a sensory organ composed of 12 ciliated neurons and two glial cells. This organ serves as a model to explore how neurons and glia communicate in the context of tissues within a living animal.Cilia are sensory projections present on several types of cells and used to detect external stimuli. The ciliated sensory neurons of C. elegans have been widely used as a model to understand cilia biology. Ciliary traffic homeostasis is key in cilia biology, and several diseases, collectively referred to as ciliopathies, are caused by dysfunctional cilia. In C. elegans, cilia dysfunction disrupts sensory responses to odors, food, repellent molecules, and temperature changes. In addition to their sensory function, cilia are believed to aid in the communication between cells and individuals by excreting extracellular vesicles larger than 100 nm, called ectosomes.First, I show how ectosomes bud from the ciliary membrane of ciliated neurons of hermaphroditic animals, a mechanism thought to be exclusive to the male ciliated neurons of C. elegans. To describe how and which ciliated sensory neurons produce ectosomes, I selected potential cargoes of ciliated ectosomes that were marked by transgenesis or knock-in. These new ectosome markers include tetraspanins TSP-6, 7 and 4, as well as other membrane receptors or sensory machinery proteins. Ciliary distribution and entry into ectosomes of these markers have been studied in control animals as well as in several mutants that disrupt ciliary traffic at different levels, leading to the accumulation of ciliary proteins in specific ciliary compartments as well as the production of ectosomes. I demonstrated how ciliary protein accumulation increases ectosome production, providing a mechanism to reduce the protein load within the cilium. Next, I determined that ectosomes originated from membrane budding at the tip or base of the cilium. These different origins give rise to different fates; ectosomes released from the tip of the cilium are directly released into the environment, whereas ectosomes budding at the base of the cilium are easily captured by glial cells. Interestingly, the slowing of ectosome uptake by glial cells resulted in changes in cilia shape and disrupted sensory functions. Overall, I explored how ciliated neurons increase extracellular vesicle release under conditions that mimic certain ciliopathies and the role of glia in the uptake of released material. This provides a framework in which neurons potentially participate in communication with glial cells under pathological conditions. Further experiments to prove the functional role of such communication could be important for understanding certain aspects of ciliary diseases.In parallel, but in an unrelated context, I explored the dynamics of glial cells from the same sensory organ of C. elegans. I have demonstrated how AMsh cell morphology changes upon exposure to various cellular stressors. I report how the expression of proteotoxic proteins affects the cellular shape of AMsh by increasing its surface area and length and inducing the production of cellular fragments that are sometimes released from the cell, in a mechanism reminiscent of exopher extrusion. One such cytotoxic protein used to disrupt AMsh cell morphology is mCherry. I showed that it largely accumulated in LAMP1-positive compartments, corresponding to late endosomes and/or lysosomes. I explored how mCherry enters the endolysosomal pathway and proposed that it enters the early stages of the endosomal pathway through microautophagy. However, further experiments are needed to better understand how mCherry interacts with the endolysosomal pathway and whether its presence disrupts the normal endosomal flow. Finally, I defined the association between the AMsh and its surrounding tissue, the hypodermis, showing how they grow together and the presence of close contacts that connect the two tissues. Additionally, I show how hypodermic cells can take up glia-derived cell fragments and secrete extracellular matrix factors that are important for maintaining the integrity of the nervous system.