Sphingomyelin (SM) is a major component of mammalian cell membranes. Its bulk production in the trans-Golgi provides a thermodynamic trap for cholesterol synthesized in the ER to promote the formation of a SM/sterol concentration gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that helps specify organelle identity and function. A previous study identified mutations in SM synthase SMS2 as the underlying cause of a hereditary form of osteoporosis and skeletal dysplasia. This work shows that two missense SMS2 variants linked to the most severe bone phenotype, p.I62S and p.M64R, retain full enzymatic activity but are unable to leave the ER owing to a defective autonomous ER export signal. Consequently, bulk production of SM is mistargeted to the ER, the site for de novo synthesis of the SM precursor ceramide. Combining organellar lipidomics with the application of lipid reporters, I find that cells harboring these pathogenic SMS2 variants accumulate plasma membrane-like SM levels in the ER and display a disrupted SM asymmetry at the plasma membrane, presumably due to a constitutive SM scrambling in the ER. These aberrant SM distributions also occur in patient-derived fibroblasts and are accompanied by significant imbalances in cholesterol organization and lipid order along the secretory pathway. Moreover, I find that a more common nonsense SMS2 variant associated with a milder bone phenotype, p.R50X, yields a truncated but catalytically active enzyme that is mistargeted to an early Golgi compartment. Collectively, these data indicate that pathogenic SMS2 variants undermine the capacity of cells to uphold nonrandom lipid distributions in the secretory pathway that may be critical for the bone forming activity of osteogenic cells.

L'organisation orientationnelle bio-moléculaire des lipides et des protéines dans la membrane plasmique constitue un facteur important dans les processus biologiques au cours desquelles les fonctions peuvent être reliées aux mécanismes d'orientation et d'organisation. Le concept de séparation transitoire des phases à l'échelle nanométrique dans les domaines ordonnés et désordonnés, aussi appelé « radeau lipidique », est maintenant largement accepté. De plus, les domaines ordonnés contiennent des protéines de signalisation, ce qui souligne l'importance des séparations de phase au cours des processus de signalisation. Dans cette thèse de doctorat, nous avons étudié l'ordre orientationnel moléculaire de la protéine de signalisation MHC Class I et de reporters lipidiques tels que di-8-ANEPPQ et DiI(C18). Nous avons étudié l'ordre orientationnel moléculaire de la protéine de signalisation MHC Class I et des reporters lipidiques par imagerie d'anisotropie de fluorescence résolue en polarisation. Nous avons observé l'influence du cytosquelette d'actine sur l'ordre orientationnel moléculaire de la protéine MHC et des reporters lipidiques dans la membrane plasmique. De plus, nous avons trouvé que l'ordre orientationnel moléculaire des reporters dépend de la morphologie cellulaire. Nous avons examiné les plis membranaires en modifiant la forme des cellules de façon mécanique ou pharmacologique. / Biomolecular orientational organization of lipids and proteins in the plasma membrane is a crucial factor in biological processes where functions can be closely related to orientation and ordering mechanisms. The concept of transient nanosized phase separations in ordered and disordered domains, called "lipid rafts" is now widely accepted. Furthermore, the ordered domains are enriched in signaling proteins, which highlights the crucial impact of phase separation during the signaling processes. While this field has been so far largely addressed by studying the translational diffusion behavior of membrane proteins and lipid reporters by Single Molecule Tracking (SMT) or Fluorescence Correlation Spectroscopy (FCS), only little is known about the orientational behavior of signaling proteins and lipid reporters in the plasma membrane. In this PhD thesis we investigated the molecular orientational order of the signaling molecule MHC Class I protein using fluorescence anisotropy imaging as well as of lipid reporter di-8-ANEPPQ using polarization-resolved fluorescence imaging. Fluorescence anisotropy imaging requires a fluorescent label rigidly attached to the system under study, able to report its orientational order behavior. Thus, MHC Class I protein has been successfully labeled in a rigid way. We analyzed the orientational order of MHC Class I protein quantitatively in the endomembrane and plasma membrane and we found that the orientational order of MHC Class I protein in both membranes depends primarily on the maturation state of the protein and its interaction with the cytoskeleton.