Calcium-dependent translocation of S100A11 requires tubulin filaments.

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Reconstituted P2/myelin-lipid multilayers.

A complex forms when bovine P2 protein is added to single-bilayer vesicles created by sonicating myelin lipids. The complex was studied by biochemical analysis, freeze-fracture (FF) and thin-section electron microscopy (EM), and by X-ray diffraction. Smaller amounts of P2 cause the vesicles to aggregate and fuse whereas larger amounts (greater than or equal to 4 wt%) cause multilayers to form. Binding saturates at 15 wt% P2. FF EM shows that large, flat multilayers form within 15 min of addition of P2. Only smooth fracture faces are seen, as expected for a peripheral membrane protein. X-ray diffraction shows a constant repeating distance in the multilayers: 86.0 +/- 0.7 A between the centers of bilayers in the range 4 wt% less than or equal to P2/(P2 + lipid) less than or equal to 15 wt%. Assuming a 53 A-thick bilayer, the space between bilayers is 33 A wide. This is a wider space than for myelin basic protein (MBP) (20-25 A wide). The respective widths are consistent with a compact, globular structure for P2 and a flattened shape for MBP. Calculated electron-density profiles of the lipids with and without P2 reveal the protein largely in the interbilayer spaces, with a small part possibly inserted into the lipid headgroup layers. The different proportions of P2 in the sciatic nerve of various species are tentatively correlated with the different average widths observed by X-ray diffraction for the cytoplasmic space (major period line) between bilayers in the respective sciatic myelins.

Cholesterol-phosphatidylcholine interactions in multilamellar vesicles.

We have investigated the phase behavior of dipalmitoylphosphatidylcholine-cholesterol bilayers using both the fluorescence of bilayer-associated 1,6-diphenyl-1,3,5-hexatriene (DPH) and freeze-fracture electron microscopy to elucidate specimen structure. Arrhenius analysis of the fluorescence-derived "microviscosity" parameter reveals temperature-induced structural changes in these membranes. In addition, isotherms of DPH fluorescence anisotropy and total intensity are used to detect alterations in membrane structure with varying cholesterol content. Freeze-fracture electron microscopic studies, utilizing rapid "jet-freezing" techniques, show strikingly different fracture-face morphologies for different combinations of sample cholesterol content and temperature. A phase diagram is proposed that offers a unifying interpretation of the fluorescence and freeze-fracture results. In this interpretation, inflections in temperature-scanning and isothermal fluorescence measurements reveal phase lines in the dipalmitoylphosphatidylcholine-cholesterol membranes Two-phase regions of the proposed phase diagram correspond to samples showing two coexisting fracture-face morphologies, while single-phase regions produce membranes having only one clearly identifiable structure. The proposed phase diagram provides an explanation for several conflicting literature proposals of stoichiometries for phosphatidylcholine-cholesterol complexes in membranes. These stoichiometric complexes correspond to the boundaries of two-phase areas in the gel region of the phase diagram. To better approximate the effect of cholesterol on natural membranes, the structure of egg phosphatidylcholine-cholesterol multilamellar vesicles was also investigated by using DPH fluorescence. The results for this complex natural phospholipid system are interpreted by comparison with the synthetic phospholipid results.

Acyl chain order and lateral domain formation in mixed phosphatidylcholine--sphingomyelin multilamellar and unilamellar vesicles.

The phase behavior of mixtures of dimyristoylphosphatidylcholine (DMPC) with N-palmitoylsphingosinephosphorylcholine (C16SHP) has been investigated in both small unilamellar and large multilamellar vesicles. The steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) has been used to detect temperature-induced structural changes in these membranes. In addition, electron microscopy has revealed vastly different fracture-face morphologies for large multilamellar vesicles "jet-frozen" from different temperatures. These data have been interpreted in terms of proposed phase diagrams for this lipid mixture. The shapes of the proposed phase diagrams have led us to conclude that phosphatidylcholine and sphingomyelin species of similar acyl chain length mix freely in both highly curved and uncurved bilayers, except at temperatures at which both lipids are in low-temperature, ordered phases. In addition, the similarity of these phase diagrams to phase diagrams for analogous mixtures of pure phosphatidylcholines suggested that sphingomyelin and phosphatidylcholine suggested that sphingomyelin and phosphatidylcholine species might substitute for each other in supporting the lamellar phase necessary for each other in supporting the lamellar phase necessary to cell membrane structure. Finally, the anisotropy of DPH fluorescence was found to be essentially invariant with sphingomyelin content at temperatures just above and below the solid--liquid phase separation in small unilamellar vesicles. This demonstrates that the sphingomyelin backbone, per se, does not order the membrane bilayer. These results are discussed in terms of the possible role of sphingomyelin in controlling acyl chain order within mammalian cell membranes.

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum.

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.

Phase behavior of mixed phosphatidylglycerol/phosphatidylcholine multilamellar and unilamellar vesicles.

The phase behavior of dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) mixtures has been studied in both small, unilamellar vesicles and large, multilamellar vesicles. We have used both the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and high-sensitivity differential scanning calorimetry to detect temperature-dependent changes in membrane structure. Electron microscopy has demonstrated different fracture face morphologies for large, multilamellar vesicles depending on sample composition and temperature. These data have been interpreted in terms of proposed phase diagrams for this lipid mixture. The shapes of the proposed phase diagrams have led us to conclude that DMPC and DC15PG mix freely in the plane of a lipid bilayer only at less than 50 mol % DC15PG. At higher DC15PG content, the data have been interpreted as reflecting substantial compositional inhomogeneities in the plane of the bilayer, if not phase immiscibility, even in the fluid phase. In addition, small vesicles containing greater than 50 mol % DC15PG were unstable in the ordered phase and spontaneously converted to larger vesicles. Finally, the anisotropy of DPH fluorescence was found to be invariant with DC15PG content at temperatures just above the liquidus phase line in small, unilamellar vesicles. This demonstrated that inclusion of negatively charged phosphatidylglycerol does not noticeably affect the order within the acyl chain region of the bilayer, relative to phosphatidylcholine.

Relationship between lateral diffusion, collision frequency, and electron transfer of mitochondrial inner membrane oxidation-reduction components.

Fluorescence recovery after photobleaching was used to determine the diffusion coefficients of the oxidation-reduction (redox) components ubiquinone, complex III (cytochromes b-c1), cytochrome c, and complex IV (cytochrome oxidase) of the mitochondrial inner membrane. All redox components diffuse in two dimensions as common-pool electron carriers. Cytochrome c diffuses in two and three dimensions concomitantly, and its diffusion rate, unlike that of all other redox components, is modulated along with its activity by ionic strength. The diffusion coefficients established in this study reveal that the theoretical diffusion-controlled collision frequencies of all redox components are greater than their experimental maximum (uncoupled) turnover numbers. Since electron transport is slower than the theoretical limit set by the lateral diffusion of the redox components, ordered chains, assemblies, or aggregates of redox components are not necessary to account for electron transport. Rather, mitochondrial electron transport is diffusion coupled, consistent with a "random-collision model" for electron transport.

Expression of members of the multidrug resistance protein family in human term placenta.

The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta-glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.