Non-senescent keratinocytes organize in plasma membrane submicrometric lipid domains enriched in sphingomyelin and involved in re-epithelialization

Abstract

Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.

Spatial Relationship and Functional Relevance of Three Lipid Domain Populations at the Erythrocyte Surface

“…super resolution microscopy or fluorescence lifetime spectroscopy) [13][14][15][16] and more relevant lipid probes [17,18] have allowed to evidence bigger (submicrometric instead of nanometric) and more stable lipid domains. Those have been observed on prokaryotic cells [19], yeast [20,21] and various eukaryotic cells like keratinocytes [22], fibroblasts [23] and RBCs [24][25][26]. Focus is generally made on sphingolipids and sterols as they are two major lipids of the PM external leaflet of many cells and because they are known to be enriched in rafts and associated with membrane fluidity regulation.…”

Assessing the Photoprotective Effects of Fluorescent Sphingomyelin Against UVB Induced DNA Damage in Human Keratinocytes

“…The signal obtained was predominantly concentrated around the plasma membrane [65]. The heterogeneity of lysenin staining was also described and the overall signal declined as the cells reached senescence [65].…”

“…Colocalization of lysenin with FSM signal was quantified, and Mound demonstrated that lysenin signal diminished after the addition of SMase, an enzyme that degrades SM [65]. The signal obtained was predominantly concentrated around the plasma membrane [65]. The heterogeneity of lysenin staining was also described and the overall signal declined as the cells reached senescence [65].…”

“…This protein binds selectively to SM, as demonstrated by ELISA, TLC, and other methods [64]. Mound et al used lysenin, in conjunction with FSM, to visualize lipid raft organization in the plasma membrane of KRTs [65]. Colocalization of lysenin with FSM signal was quantified, and Mound demonstrated that lysenin signal diminished after the addition of SMase, an enzyme that degrades SM [65].…”

“…Mound et al used lysenin, in conjunction with FSM, to visualize lipid raft organization in the plasma membrane of KRTs [65]. Colocalization of lysenin with FSM signal was quantified, and Mound demonstrated that lysenin signal diminished after the addition of SMase, an enzyme that degrades SM [65]. The signal obtained was predominantly concentrated around the plasma membrane [65].…”