A non-exchangeable fluorescent phospholipid analog as a membrane traffic marker of the endocytic pathway

The fluorescent phospholipid analog N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE) was inserted into the plasma membrane of Baby hamster kidney cells at low temperature (2 degrees C). The mobility characteristics of the analog--as revealed by fluorescence photobleaching recovery--were very similar to those of membrane-inserted 1-acyl-2[6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]caproyl] phosphatidylcholine (C6-NBD-PC). Upon warming to 37 degrees C, followed by a 1-h incubation, all N-Rh-PE was located intracellularly. By contrast, after the same time interval, approximately 10% of the cell-associated PC-derivative was found intracellularly. Furthermore, the analogs moved to different intracellular sites, as N-Rh-PE associates with perinuclear and peri-Golgi structures, whereas C6-NBD-PC appears mainly in the Golgi complex. Colocalization with organelle-specific probes and Percoll gradient analysis identified the N-Rh-PE-labeled structures as lysosomes. Temperature and energy-dependent experiments supported the endocytic pathway as the mechanism of N-Rh-PE internalization. The mechanism of N-Rh-PE internalization appears to differ from that of C6-NBD-PC. In conjunction with a difference in the efficiency of removal of the lipid derivatives from the plasma membrane, the results suggest that N-Rh-PE is selectively internalized, implying that sorting of the lipid analogs already occurs at the level of the plasma membrane. The distinct difference in physical appearance of the probes after membrane insertion, i.e., N-Rh-PE being present as small clusters and C6-NBD-PC as monomers, could explain the selective sorting and internalization of N-Rh-PE. The results demonstrate that N-Rh-PE may serve as a useful marker for studying membrane traffic during endocytosis.     

Comparative study of the lateral motion of extrinsic probes and anthracene-labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non-fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5-(N-hexadecanoyl)-aminofluorescein, 12-(9-anthroyloxy)-stearic acid, 9-(2-anthryl)-nonanoic acid and a synthetic anthracene-phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 - 2 x 10(-9) cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene-labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.     

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.     

Analysis of the distribution of cholesterol in the intact cell

We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.     

Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD- PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD- PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.     

Reduction of sphingomyelin level without accumulation of ceramide in Chinese hamster ovary cells affects detergent-resistant membrane domains and enhances cellular cholesterol efflux to methyl-beta -cyclodextrin

We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was approximately 40 and approximately 15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [(3)H]cholesterol showed that the [(3)H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-beta-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains.     

Differences in the Transbilayer and Lateral Motions of Fluorescent Analogs of Phosphatidylcholine and Phosphatidylethanolamine in the Apical Plasma Membrane of Bovine Aortic Endothelial Cells

In the plasma membrane of various eucaryotic cell types, in particular blood platelets and erythrocytes, it is known that phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer and that this transverse asymmetry is controlled by an aminophospholipid translocase activity. In this respect, it was of interest to check whether there are differential transbilayer movements between amino- and neutral phospholipids in the apical plasma membrane of vascular endothelial cells which form the inner nonthrombogenic lining of the large blood vessel. In the first step we compared the transbilayer localization and also the rate of lateral motion of two fluorescent analogs of phosphatidylcholine and phosphatidylethanolamine, namely C6-NBD-PC and C6-NBD-PE, inserted into the apical plasma membrane of bovine aortic endothelial cells, in vitro. By the use of back-exchange experiments we have found that C6-NBD-PC could be removed from the cell membrane toward the culture medium regardless of the incubation conditions used, i.e., just after cell labeling at 0°C or even after further cell incubation for 1 h at 0 or 20°C. In contrast, C6-NBD-PE could be removed only when the cells were maintained at 0°C. After incubation for 1 h at 20°C, 85% of the probe molecules remained nonexchangeable, indicating probe translocation from the outer to the inner leaflet of the lipid bilayer. This "flip" process, which occurred at 20°C, was abolished when the endothelial cells were preincubated with N-ethylmaleimide, diamide, vanadate (VO^3-₄) and vanadyl (VO²⁺) ions, a set of substances which inhibit aminophospholipid translocase activity in various systems, and with a combination of sodium azide and 2-deoxyglucose which led to nearly complete ATP depletion in the cells. Fluorescence recovery after photobleaching experiments were also carried out to specify more precisely the localization and dynamics of the probes in the two leaflets of the plasma membrane lipid bilayer. They produced lateral diffusion coefficients D of 1.2 ± 0.05 × 10^-9 cm²/s for C6-NBD-PC and 2.8 ± 0.3 × 10^-9 cm²/s for C6-NBD-PE, when the two probes were located in the outer leaflet of the plasma membrane, just after cell labeling at 0°C. After cell incubation for one hour at 20°C, i.e., when C6-NBD-PC was still in the outer leaflet whereas C6-NBD-PE was translocated in the inner leaflet, D was observed to slightly increase for C6-NBD-PC (D = 1.9 ± 0.06 × 10^-9 cm²/s) and to greatly increase by at least a factor of 3 for C6-NBD-PE (D = 9.1 ± 0.9 × 10^-9 cm²/s). These results show that the plasma membrane of bovine aortic endothelial cells is equipped with a protein-dependent and energy-mediated phosphatidylethanolamine translocase activity and that the lateral diffusion rate of this phospholipid is much faster in the inner than in the outer leaflet of the lipid bilayer, thus indicating large differences in the fluidity of the two halves of this membrane.     

Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts

The kinetics of low density lipoprotein (LDL) cholesterol transport to the plasma membrane of Chinese hamster ovary (CHO) cells was studied. LDL was reconstituted with [3H]cholesteryl linoleate and added to CHO cells in a pulse-chase experiment. The internalization and lysosomal cleavage of reconstituted LDL (rLDL) [3H]cholesteryl linoleate to free [3H]cholesterol occurred with a half-time of 37 min after a 30-min lag. The rate of transport of released [3H]cholesterol to the plasma membrane was measured by brief (20-30 sec) cholesterol oxidase treatment of intact, adherent cells: the half-time of transport was 42 min. The similarity in the rate of free cholesterol release from rLDL and transport of this cholesterol to the plasma membrane suggests very rapid transport of rLDL cholesterol from the lysosome to the plasma membrane. Cells were shown to be intact throughout the cholesterol oxidase treatment by the absence of cell-derived lactate dehydrogenase (LDH) activity or K+ in the assay buffer.     

Topographical distribution of a membrane-inserted fluorescent phospholipid analogue during cell fusion

Potential alterations in the transbilayer distribution of lipid molecules during cell-cell fusion were studied, using the fluorescent phospholipid analogue 1-acyl, 2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC). The fluophore was inserted into the outer leaflet of the plasma membrane of Chinese hamster fibroblasts from an exogenous source and cell-cell fusion was induced either with Sendai virus or polyethylene glycol (PEG). After fusion, the cells were examined under a fluorescence microscope and the pool of tagged lipid molecules in the external monolayer was determined quantitatively. The results showed that in contrast to PEG-induced cell fusion, substantial redistribution of the lipid marker occurred when cell fusion was induced by Sendai virus and it was estimated that approx. 40% of exogenously supplied lipid was internalized. The possible mechanism causing lipid redistribution in the case of Sendai virus-induced cell fusion is discussed.     

Parameters affecting low-pH-mediated fusion of liposomes with the plasma membrane of cells infected with influenza virus

Unilamellar liposomes can be fused at low pH with the plasma membrane of cells that express the hemagglutinin glycoprotein of influenza virus on their surface [van Meer, G., & Simons, K. (1983) J. Cell Biol. 97, 1365-1374]. Here, we have resolved this fusion process into two kinetically distinct steps. The first and more rapid step converts the bound liposome to a form that can no longer be released by neuraminidase. The second step is the actual membrane fusion as measured by the loss of resonance energy transfer between two liposomal fluorescent phospholipids, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolami ne (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In contrast to the first step, the rate of the second one was highly dependent on the liposomal lipid composition and the cell type used. The replacement of 50% of the phosphatidylcholine (PC) in egg PC-cholesterol liposomes by unsaturated phosphatidylethanolamine (PE) species increased the rate of fusion at least 2-fold. Of the PE-containing liposomes that were associated with Madin-Darby canine kidney (MDCK) cells after 30 s of fusion, 80% had actually fused with the plasma membrane. Fringe pattern fluorescence photobleaching experiments showed that after fusion a fraction of the cell-associated N-Rh-PE diffused laterally in the plasma membrane. Without fusion, the N-Rh-PE was completely immobile. Under optimal conditions, the mobile fractions were 65% on MDCK cells and 78% on baby hamster kidney cells. The mobility was acquired simultaneously with the dilution of the fluorescent phospholipids as measured from the loss of resonance energy transfer. The mobile fraction of N-Rh-PE on the cell surface can therefore be used as a second independent measure of actual membrane fusion. Finally, we observed that upon fusion up to 80% of the nonexchangeable liposome markers cholesterol [14C]oleate and glycerol tri[14C]oleate became accessible to cellular hydrolases. The results showed that this hydrolysis assay can also be used to monitor the second step of the fusion process.     

Diffusional mobility of the cystic fibrosis transmembrane conductance regulator mutant, delta F508-CFTR, in the endoplasmic reticulum measured by photobleaching of GFP-CFTR chimeras

Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.     

Domains responsible for the differential targeting of glucose transporter isoforms.

Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. In insulin-responsive tissues such as adipose tissues and muscle, GLUT4 protein resides mainly in the intracellular region in a basal condition and is translocated to the plasma membrane upon stimulation of insulin. In contrast, GLUT1 protein was distributed about equally between plasma membranes and low density microsomal membranes in 3T3-L1 adipocytes. Furthermore, GLUT1 and GLUT4 were reported to be differentially targeted to the plasma membrane and intracellular region, respectively, when expressed in Chinese hamster ovary cells and HepG2 cells. To elucidate the differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1 have been stably expressed in Chinese hamster ovary cells. Immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity revealed that two domains of GLUT4, which are not the NH2- or COOH-terminal domain, determine its targeting to the intracellular vesicles. The first domain contains the consensus sequence of the leucine zipper structure, suggesting that a dimer-forming structure of the glucose transporter might be required for its proper targeting. The other domain contains 28 amino acids, nine of which are different between GLUT1 and GLUT4. Immunoelectron microscopy revealed that the chimeric transporters containing both of these two domains of GLUT1, only the first domain of GLUT1, and none of the domains, exhibited a different cellular distribution with approximately 65, 30, and 15% of the transporters apparently on the plasma membrane, respectively. The addition of insulin did not alter the apparent cellular distributions of these chimeric transporters. These domains would be specifically recognized by intracellular targeting mechanisms in Chinese hamster ovary cells.     

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.     

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.

Plasma membranes were prepared from cultured Chinese hamster ovary cells utilizing a two-phase polymer system and were characterized by enzymatic and chemical assay, and by electron microscopy. The usual degree of purification of presumptive membrane markers such as Na+-K+ ATPase (ATP phosphohydrolase, EC 3.6.1.3) ranged from three-to eightfold. Gel electrophoresis in SDS revealed several polypeptides and two glycopeptides which were enriched in the plasma membrane fraction.     

Incomplete functional differentiation of HL-60 leukemic cells by synthetic lipopeptides. Partial inhibition by pertussis toxin of enhanced superoxide formation.

In human neutrophils, the synthetic lipopeptide, N-palmitoyl-S-[2,3- bis(palmitoyloxy-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-( S)-lysyl-(S) -lysyl-(S)-lysine [Pam3CysSer(Lys)4], activates NADPH-oxidase catalyzed superoxide (O2-) formation through pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms (Seifert, R., Schultz, G., Richter-Freund, M., Metzger, J., Wiesmüller, K.-H., Jung, G., Bessler, W. G. & Hauschildt, S. (1990) Biochem. J. 267, 795-802). We studied the effects of lipopeptides on differentiation of HL-60 leukemic cells. Pam3CysSer(Lys)4 enhanced phorbol-12-myristate-13-acetate-induced O2- formation (presumably through the expression of components of NADPH oxidase) in a concentration-dependent manner with a half-maximal effect at 100 ng/ml and a maximum at 1 microgram/ml. The effect of the lipopeptide was evident after 24 h and reached a plateau after 48 h. (2S,6S)-2-Palmitoylamino-6,7- bis(palmitoyloxy)heptanoyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S) -lysyl-(S)-lysine enhanced O2- formation as well. The effects of Pam3CysSer(Lys)4 were potentiated by dibutyryl cAMP, dimethyl sulfoxide, retinoic acid, 1,25-dihydroxyvitamin D3, interferon-gamma and tumor-necrosis-factor-alpha. Pertussis toxin, but not its B-oligomer, partially inhibited enhanced O2- formation induced by Pam3CysSer(Lys)4. O2- formation induced by arachidonic acid and gamma-hexachlorocyclohexane were more sensitive to inhibition by pertussis toxin than O2- formation induced by phorbol 12-myristate 13-acetate. Enhanced O2- formation induced by dibutyryl cAMP was not affected by pertussis toxin. Unlike ATP, histamine, prostaglandin E1 and the beta-adrenergic agonist, isoproterenol, Pam3CysSer(Lys)4 did not increase cytosolic Ca2+ [( Ca2+]i) in undifferentiated HL-60 cells. Histamine but not lipopeptides stimulated high-affinity GTPase of guanine-nucleotide-binding proteins in membranes of undifferentiated HL-60 cells. In Pam3CysSer(Lys)4-differentiated HL-60 cells, the responsiveness to the [Ca2+]i-increasing agonists, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, C5a and leukotriene B4, was increased, whilst the responsiveness to prostaglandin E1 and isoproterenol was decreased. Pam3CysSer(Lys)4 did not inhibit proliferation of HL-60 cells but decreased transferrin receptor expression and increased C3bi receptor expression. Pertussis toxin did not affect proliferation and expression of transferrin and C3bi receptors. Dibutyryl cAMP was considerably more effective than Pam3CysSer(Lys)4 at inducing alterations in the above parameters. Our results suggest that (a) Pam3CysSer(Lys)4 induces incomplete functional differentiation of HL-60 cells through a mechanism which does not depend on a rise in [Ca2+]i and is different from that of other differentiation-inducing substances and (b) the mechanism by which Pam3CysSer(Lys)4 induces differentiation involves pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms.     

Amyloid beta peptide 1-40 enhances the action of Toll-like receptor-2 and -4 agonists but antagonizes Toll-like receptor-9-induced inflammation in primary mouse microglial cell cultures

The interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells and macrophages after application of defined Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) (TLR4), the synthetic lipopeptide Pam3Cys-Ser-Lys4 (Pam3Cys) (TLR2) and single-stranded unmethylated CpG-DNA (CpG) (TLR9)] alone and in combination with amyloid beta peptide (Abeta) 1-40. Abeta1-40 stimulated microglial cells and macrophages primed by interferon-gamma in a dose-dependent manner. Co-administration of Abeta1-40 with LPS or Pam3Cys led to an additive release of nitric oxide (NO) and tumour necrosis factor alpha (TNF-alpha). This may be one reason for the clinical deterioration frequently observed in patients with Alzheimer's disease during infections. In contrast, co-application of Abeta1-40 with CpG led to a substantial decrease of NO and TNF-alpha release compared with stimulation with CpG alone. Abeta1-40 and CpG did not co-localize within the same subcellular compartment, making a direct physicochemical interaction as the cause of the observed antagonism very unlikely. This suggests that not all TLR agonists enhance the stimulatory effect of A beta on innate immunity.     

Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin

Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells.     

Intracellular transport of phosphatidylcholine to the plasma membrane

We have used pulse-chase labeling of Chinese hamster ovary cells with choline followed by plasma membrane isolation on cationic beads to study the transport of phosphatidylcholine from the endoplasmic reticulum to the plasma membrane. We have found that the process is rapid (t1/2 [25 degrees C] = 2 min) and not affected by energy poisons or by cytochalasin B, colchicine, monensin, or carbonyl cyanide p-chlorophenylhydrazone. Cooling cells to 0 degree C effectively stops the transport process. The intracellular transport of phosphatidylcholine is distinct in several ways from the intracellular transport of cholesterol (Kaplan, M. R., and R. D. Simoni, 1985, J. Cell. Biol., 101:446-453).     

Influence of alcohols, temperature, and region on the mobility of lipids in neuronal membrane

Fluorescence recovery after photobleaching was used to examine lipid diffusibility in different regions of Aplysia neurons. Differences in diffusion of 1-acyl-2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)]aminohexanoyl)phosphatidylcholine (NBD-C6-PC) in the cell body, axon hillock, and axon were not apparent. Lipid diffusibility during temperature variations and exposure to alcohols was also examined by photobleaching techniques. For these studies, all measurements were made on the cell body. Alcohols were found to be selective in their effects upon the diffusibility of lipid probes. Neither ethanol nor butanol affected the diffusibility of NBD-PC. However, at the same concentrations, both of these alcohols caused a significant increase in the diffusion coefficient (D) for rhodamine-phosphatidylethanolamine (Rho-PE). The diffusion coefficient for NBD-PC in the cell body plasma membrane did not increase with warming, between 4 degrees C and 25 degrees C. The fraction of lipid probe free to diffuse (per cent recovery; %R) however, increased as temperature increased, within this range. The nonconventional relationship between temperature and D was even more pronounced for Rho-PE. As temperature increased, D became smaller for this probe, concurrent with an increase in %R. These results suggest that immobile viscous lipid is recruited into a mobile fraction as temperature increases, resulting in the maintenance of constant diffusibility. The effects of temperature on D and %R, and the selective effects of alcohols on lipid diffusibility suggest that the membrane is heterogeneously organized, on a submicroscopic scale, into domains. The implications of this organization for nerve function and responses of nervous systems to temperature and anesthetics are discussed.     

Isolation and preliminary characterization of the Chinese hamster thymidine kinase gene.

The Chinese hamster thymidine kinase (TK) gene has been isolated from a recombinant phage library constructed with genomic DNA from mouse Ltk- cells transformed to Tk+ by transfection with Chinese hamster genomic DNA. The phage library was screened by the Benton-Davis plaque hybridization technique, using as probes, subclones of recombinant phage that were isolated from mouse Ltk+ transformants by the tRNA suppressor rescue method. The Chinese hamster TK gene is contained within 13.2 kilobases of genomic DNA in the isolate designated lambda 34S4. This gene, defined by restriction enzyme sensitivity experiments, homology studies with the chicken TK gene, and mRNA blotting experiments, may extend over 8.5 kilobases. Subclones of the lambda 34S4 isolate used as hybridization probes identified a 1,400-nucleotide polyadenylated RNA as the hamster TK mRNA. The abundance of this mRNA varies dramatically in Chinese hamster cells cultured under various growth conditions, providing direct evidence that the growth dependence of TK activity may be regulated in an important way at the level of cytoplasmic TK mRNA.