Ultrastructure and lipid composition of detergent-resistant membranes derived from mammalian sperm and two types of epithelial cells.

Lipid rafts are micro-domains of ordered lipids (Lo phase) in biological membranes. The Lo phase of cellular membranes can be isolated from disordered lipids (Ld phase) after treatment with 1 % Triton X-100 at 4 °C in which the Lo phase forms the detergent-resistant membrane (DRM) fraction. The lipid composition of DRM derived from Madin-Darby canine kidney (MDCK) cells, McArdle cells and porcine sperm is compared with that of the whole cell. Remarkably, the unsaturation and chain length degree of aliphatic chains attached to phospholipids is virtually the same between DRM and whole cells. Cholesterol and sphingomyelin were enriched in DRMs but to a cell-specific molar ratio. Sulfatides (sphingolipids from MDCK cells) were enriched in the DRM while a seminolipid (an alkylacylglycerolipid from sperm) was depleted from the DRM. Treatment with <5 mM methyl-ß-cyclodextrin (MBCD) caused cholesterol removal from the DRM without affecting the composition and amount of the phospholipid while higher levels disrupted the DRM. The substantial amount of (poly)unsaturated phospholipids in DRMs as well as a low stoichiometric amount of cholesterol suggest that lipid rafts in biological membranes are more fluid and dynamic than previously anticipated. Using negative staining, ultrastructural features of DRM were monitored and in all three cell types the DRMs appeared as multi-lamellar vesicular structures with a similar morphology. The detergent resistance is a result of protein-cholesterol and sphingolipid interactions allowing a relatively passive attraction of phospholipids to maintain the Lo phase. For this special issue, the relevance of our findings is discussed in a sperm physiological context.

Erythrophagocytosis by angiogenic endothelial cells is enhanced by loss of erythrocyte deformability.

OBJECTIVE: Angiogenic endothelial cells can function as phagocytes, and phagocytosis is initiated via the opsonin lactadherin. In this study, we examined the interaction between lactadherin-opsonized erythrocytes with reduced deformability and angiogenic endothelium, as loss of deformability is characteristic for suicidal and aged erythrocytes. MATERIALS AND METHODS: We used the Arg-Gly-Asp (RGD)-modified erythrocyte model and investigated the deformability parameter by cross-linking erythrocyte membranes through treatment with glutaraldehyde. Association in vitro with primary endothelial cells was detected by flow cytometry and visualized by light, fluorescent, and electron microscopy. Involvement of two crucial factors in phagocytosis, alpha(v)-integrins and Rho guanosine triphosphatase family member Rac1, was studied using small interfering RNA technology. Modified erythrocytes were administered in vivo into tumor-bearing mice to detect phagocytosis by endothelial cells. RESULTS: Glutaraldehyde-treated (rigid) RGD-modified erythrocytes showed a strongly enhanced endothelial cell association compared to flexible RGD-modified erythrocytes. Knockdown by small interfering RNA lipoplexes of alpha(v)-integrins and Rac1 confirmed classical tethering and internalization of rigid RGD-erythrocytes. Upon in vivo administration, tumor endothelium showed pronounced erythrophagocytosis. CONCLUSION: The pronounced phagocytosis of opsonized erythrocytes with reduced deformability by angiogenic growth factor-activated endothelial cells evokes new insights in endothelial cell function and suggests a role for these endothelial cells in (hematological) disorders because of their capacity to clear disordered erythrocytes.

Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells.

Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of alpha(v)-integrins. Here, we show that the overexpression of alpha(v)-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding ("tethering") and uptake ("tickling") of lactadherin (also termed MFG-E8)--opsonized particles. Phosphatidylserine (PS)--exposing multilamellar vesicles, "aged" erythrocytes, and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonization in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides--mimicking lactadherin opsonization--we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro, this was confirmed by cytochalasin D preincubation. When RGD-erythrocytes were administered intravenously in tumor-bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonized large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.

Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking.

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, beta-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.