Antoine Cominelli

Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane.

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon SM depletion by SMase. The 125I-lysenin* binding isotherm indicated saturation at 3.5 × 106 molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled submicrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.

Matrix metalloproteinase-27 is expressed in CD163+/ CD206+ M2 macrophages in the cycling human endometrium and in superficial endometriotic lesions

Matrix metalloproteinases (MMPs) are key enzymes involved in extracellular matrix remodelling. In the human endometrium, the expression and activity of several MMPs are maximal during the menstrual phase. Moreover, MMPs are thought to be involved in the pathogenesis of endometriosis and cancers, in particular with invasion and metastasis. We recently reported that MMP-27 is a unique MMP with an intracellular retention motif. We investigated the expression and cellular localization of MMP-27 in the cycling human endometrium and in endometriotic lesions. MMP-27 mRNA was detected throughout the menstrual cycle. Despite large interpatient variations, mRNA levels increased from the proliferative to the secretory phase, to peak during the menstrual phase. MMP-27 was immunolocalized in large isolated cells scattered throughout the stroma and around blood vessels: these cells were most abundant at menstruation and were identified by immunofluorescence as CD45(+), CD163(+) and CD206(+) macrophages. CD163(+) macrophages were also abundant in endometriotic lesions, but showed different patterns in ovarian or peritoneal endometriotic lesions (co-labelling for CD206 and MMP-27) and rectovaginal lesions (no co-labelling). In conclusion, MMP-27 is expressed in a subset of endometrial macrophages related to menstruation and in ovarian and peritoneal endometriotic lesions.

Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation.

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

Acute ligand-independent Src activation mimics low EGF-induced EGFR surface signalling and redistribution into recycling endosomes

Src, a non-receptor tyrosine kinase, is a key signal transduction partner of epidermal growth factor (EGF) receptor (EGFR). In human breast cancer, EGFR and Src are frequently over-expressed and/or over-activated. Although reciprocal activation is documented, mechanisms underlying Src:EGFR interactions are incompletely understood. We here exploited ts/v-Src thermo-activation in MDCK monolayers to test whether acute Src activation impacts on signalling and trafficking of non-liganded EGFR. We found that thermo-activation caused rapid Src recruitment to the plasma membrane, concomitant association with EGFR, and its phosphorylation at Y845 and Y1173 predominantly at the cell surface. Like low EGF concentrations, activated Src (i) decreased EGF surface binding without affecting the total EGFR pool; (ii) triggered EGFR endocytosis via clathrin-coated vesicles; (iii) and led to its sequestration in perinuclear/recycling endosomes with avoidance of multivesicular bodies and lysosomal degradation. Combined Src activation and EGF were synergistic for EGFR-Y845 and -Y1173 phosphorylation at some endosomes. We conclude that acute effects of Src in MDCK cells may mimic those of low EGF on EGFR activation and redistribution. Src:EGFR interactions may be sufficient to trigger EGFR activation and might contribute to its local signalling, without requiring either soluble extracellular signal or receptor over-expression.

A unique C-terminal domain allows retention of matrix metalloproteinase-27 in the endoplasmic reticulum

Matrix metalloproteinase‐27 (MMP‐27) is poorly characterized. Sequence comparison suggests that a C‐terminal extension (CTE) includes a potential transmembrane domain as in some membrane‐type (MT)‐MMPs. Having noticed that MMP‐27 was barely secreted, we investigated its subcellular localization and addressed CTE contribution for MMP‐27 retention. Intracellular MMP‐27 was sensitive to endoglycosidase H. Subcellular fractionation and confocal microscopy evidenced retention of endogenous MMP‐27 or recombinant rMMP‐27 in the endoplasmic reticulum (ER) with locked exit across the intermediate compartment (ERGIC). Conversely, truncated rMMP‐27 without CTE accessed downstream secretory compartments (ERGIC and Golgi) and was constitutively secreted. CTE addition to rMMP‐10 (a secreted MMP) caused ER retention and blocked secretion. Addition of a PKA target sequence to the cytosolic C‐terminus of transmembrane MT1‐MMP/MMP‐14 led to effective phosphorylation upon forskolin stimulation, but not for MMP‐27, excluding transmembrane anchorage. Moreover, MMP‐27 was protected from digestion by proteinase K. Finally, MT1‐MMP/MMP‐14 but neither endogenous nor recombinant MMP‐27 partitioned in the detergent phase after Triton X‐114 extraction, indicating that MMP‐27 is not an integral membrane protein. In conclusion, MMP‐27 is efficiently retained within the ER due to its unique CTE, which does not lead to stable membrane insertion. This could represent a novel ER retention system.