RESUMO
The mechanism by which poloxamer 188 (P188) seals a damaged cell membrane is examined using the lipid monolayer as a model system. X-ray reflectivity and grazing-incidence x-ray diffraction results show that at low nominal lipid density, P188, by physically occupying the available area and phase separating from the lipids, forces the lipid molecules to pack tightly and restore the barrier function of the membrane. Upon compression to bilayer equivalent pressure, P188 is squeezed out from the lipid monolayer, allowing a graceful exit of P188 when the membrane integrity is restored.
Assuntos
Lipídeos de Membrana/química , Poloxâmero/química , Difração de Raios X/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Biofísica/métodos , Membrana Celular/metabolismo , Elétrons , Bicamadas Lipídicas/química , Metabolismo dos Lipídeos , Lipídeos/química , Microscopia de Fluorescência , Modelos Estatísticos , Fosfatidilgliceróis/química , Fosfolipídeos/química , Pressão , Espalhamento de Radiação , Propriedades de Superfície , Tensoativos/química , Temperatura , Fatores de Tempo , Água/química , Raios XRESUMO
The structure of monolayers of cholesterol/ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two components within a range of compositions of cholesterol/ceramide between 100:0 and 67:33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50:50 and 30:70; between 30:70 and 0:100 only the highly crystalline phase of ceramide was detected. The latter was determined and modeled. Immunolabeling was performed with an antibody specific to the cholesterol monohydrate crystalline arrangement. The antibody recognizes crystalline cholesterol monolayers, but does not interact with crystalline ceramide. Immunofluorescence and atomic force microscopy data show that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide. As ceramide incorporates the lipid backbone common to all sphingolipids, this arrangement may be relevant to the understanding of the molecular organization of lipid rafts.
Assuntos
Ceramidas/química , Colesterol/química , Microdomínios da Membrana/química , Ar , Biofísica/métodos , Cristalização , Lipídeos/química , Microdomínios da Membrana/metabolismo , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Químicos , Modelos Estatísticos , Propriedades de Superfície , Temperatura , Água , Difração de Raios XRESUMO
Using x-ray scattering measurements we have quantitatively determined the effect of poloxamer 188 (P188), a polymer known to seal damaged membranes, on the structure of lipid monolayers. P188 selectively inserts into low lipid-density regions of the membrane and "corrals" lipid molecules to pack tightly, leading to unexpected Bragg peaks at low nominal lipid density and inducing lipid/poloxamer phase separation. At tighter lipid packing, the once inserted P188 is squeezed out, allowing the poloxamer to gracefully exit when the membrane integrity is restored.