3 resultados para Local anesthetics, Lipid membrane
em Doria (National Library of Finland DSpace Services) - National Library of Finland, Finland
Resumo:
Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology.
Resumo:
Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.
Resumo:
Cholesterol (Chol) is an important lipid in cellular membranes functioning both as a membrane fluidity regulator, permeability regulator and co-factor for some membrane proteins, e.g. G-protein coupled receptors. It also participates in the formation of signaling platforms and gives the membrane more mechanical strenght to prevent osmotic lysis of the cell. The sterol structure is very conserved and already minor structural modifications can completely abolish its membrane functions. The right interaction with adjacent lipids and the preference of certain lipid structures over others are also key factors in determining the membrane properties of cholesterol. Because of the many important properties of cholesterol it is of value to understand the forces and structural properties that govern the membrane behavior of this sterol. In this thesis we have used established fluorescence spectroscopy methods to study the membrane behavior of both cholesterol and some of its 3β-modified analogs. Using several fluorescent probes we have established how the acyl chain order of the two main lipid species, sphingomyelin (SM) and phosphatidylcholine (PC) affect sterol partitioning as well as characterized the membrane properties of 3β-aminocholesterol and cholesteryl phosphocholine. We concluded that cholesterol prefers SM over PC at equal acyl chain order, indicating that other structural properties besides the acyl chain order are important for sphingomyelin-sterol interactions. A positive charge at the 3β position only caused minor changes in the sterol membrane behavior compared to cholesterol. A large phosphocholine head group caused a disruption in membrane packing together with other membrane lipids with large head groups, but was also able to form stable fluid bilayers together with ceramide and cholesterol. The Ability of the large head group sterol to form bilayers together with ceramide was further explored in the last paper where cholesteryl phosphocholine/ceramide (Chol-PC/Cer) complexes were successfully used to transfer ceramide into cultured cells.