Description du projet
Disséquer la fusion cellulaire dans le muscle
Le muscle squelettique est constitué d’unités contractiles fonctionnelles appelées myofibres, qui émergent de la fusion de cellules musculaires, les myoblastes. La formation des myofibres nécessite le remodelage et la fusion des membranes plasmiques, des processus qui sont actuellement incomplètement caractérisés. Pour combler ce manque de connaissances, le projet MYOCLEM, financé par l’UE, développe des méthodes de microscopie électronique et optique 3D et les utilise pour élucider les acteurs moléculaires et leur fonction dans la conduite des changements associés au remodelage des membranes pendant la fusion. La connaissance du mécanisme et de la régulation de la fusion permettra non seulement d’améliorer notre compréhension de ce processus clé, mais aussi de concevoir de nouveaux traitements contre les maladies musculaires.
Objectif
Cell-to-cell fusion is a ubiquitous phenomenon essential for the physiological function of numerous tissues. A striking example is the fusion of myoblasts to form multinucleated myofibers during skeletal muscle development and regeneration. During myoblast fusion, membrane architecture must be radically remodeled. Yet, how membrane remodeling occurs on the molecular level is poorly understood as, until now, there was no approach available for visualizing dynamic changes in the cellular ultrastructure and the organization of the fusion machinery in situ.
To fill this gap, we have developed correlative light and 3D electron microscopy (CLEM) methods that allow us to identify fluorescent signals within EM samples with high sensitivity and subsequently localize the source of these signals with high precision. In this proposal, we will apply these methods in combination with live-cell imaging, biochemistry and cryo-electron tomography (ET) to deliver fundamental knowledge about the mechanism of myoblast fusion. Our specific aims are:
Aim 1: To resolve the molecular and ultrastructural events underlying cell fusion, by revealing how plasma membrane architecture is remodeled at sites of fusion using 3D EM.
Aim 2: To dissect the mechanism driving membrane remodeling during fusion, by visualizing how the fusion machinery assembles at sites of fusion and how its assembly is mirrored by changes in membrane shape, using biochemistry and live-cell imaging.
Aim 3: To determine the structure of the fusion machinery in situ, by using cryo-ET and subtomogram averaging.
Our synergetic experimental strategy will generate a quantitative, dynamic high-resolution view of the fusogenic synapse of vertebrate muscle, revealing how the fusion machinery remodels the plasma membrane at sites of fusion. These data are vital for deriving a biophysical model of myoblast fusion, understanding the general mechanism of cell fusion, and developing strategies to treat primary muscle diseases.
Champ scientifique
Not validated
Not validated
Programme(s)
Thème(s)
Régime de financement
ERC-STG - Starting GrantInstitution d’accueil
7610001 Rehovot
Israël