Microviscosity modulation during the cell cycle of neuroblastoma cells.

Microviscosity (n) of the cell membrane lipid layer was determined in synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A) by fluorescence polarization of 1,6-diphenystratum. The determined n value was maximal in mitosis, decreased markedly in the G1 phase, remained constant at a low level during the S phase, and increased again during the G2 phase. These findings imply a direct role of the cell membrane fluidity in regulation of the cell cycle.     

Lateral diffusion of lipids in complex biological membranes.

Lateral diffusion of lipids in biological membranes may be influenced by polypeptides, proteins, and other nonlipid membrane constituents. Using concepts from scaled-particle theory, we extend the free-volume model for lipid diffusion to membranes having an arbitrarily large number of components. This theory clarifies the interpretation of the free-volume theory, better reproduces the free-area dependence of lipid lateral diffusion rates, and quantitatively predicts the experimental observation that the lateral diffusion rates of membrane lipids are significantly reduced when proteins or polypeptides are incorporated in the membrane.     

Differential expression of cell cycle proteins during ageing of pancreatic islet cells

One of the major challenges for developmental biologists and investigators in the field of diabetes over the last few decades has been to dissect the origin of pancreatic endocrine cells and to accurately understand the mechanisms that regulate islet cell regeneration. While significant advances have been made recently, there continues to be a paucity of knowledge regarding the growth factor signalling pathways that directly regulate the proteins involved in islet cell cycle control. We will discuss recent work in these areas and provide insights from our studies into age-dependent alterations in the expression of growth factor signalling proteins and cell cycle proteins in islet cells.     

Theoretical comparison of the self diffusion and mutual diffusion of interacting membrane proteins.

Self diffusion and mutual diffusion in two-dimensional membrane systems are analyzed. It is shown that interprotein interactions can produce markedly different density-dependent changes in the diffusion coefficients describing these two processes; the qualitative differences are illustrated by using a theoretical formalism valid for dilute solutions. Results are obtained for three analytical potentials: hard-core repulsions, soft repulsions, and soft repulsions with weak attractions. Self diffusion is inhibited by all three interactions. In contrast, mutual diffusion is inhibited by attractions but is enhanced by repulsions. It is shown that such interaction-dependent differences in self diffusion and mutual diffusion could underlie, among other things, the disparity in protein diffusion coefficients extracted from fluorescence recovery after photobleaching and postelectrophoresis relaxation data.     

Lateral and vertical displacement of integral membrane proteins during lipid phase transition in Anacystis nidulans.

Alterations in membrane structure as a result of lipid phase transitions have been studied in Anacystis nidulans, a blue-green alga. Cells grown at 38 degrees C were subjected to temperature transitions of 38 degrees C leads to 21 degrees C and 38 degrees C leads to 10 degrees C, previously shown to produce substantial changes in photosynthetic activities, and examined by freeze-fracture electron microscopy. As a result of these treatments, large particle-free regions appeared on the fracture faces of both the plasma and thylakoid membranes. Particle density measurements suggest that the displacement of the integral membrane protein complexes occurs in both lateral and vertical directions. Returning the cells to 38 degrees C resulted in the restoration of normal membrane morphology, indicating that the proteins were not lost from the membrane. Such displacement of the integral membrane protein complexes could contribute significantly to the temperature-dependent alterations in the functional activity of membrane-bound enzymatic complexes.     

Adenylate cyclase from synchronized neuroblastoma cells: responsiveness to prostaglandin E1, adenosine, and dopamine during the cell cycle.

Neuroblastoma cells were synchronized by a combined isoleucine plus glutamine starvation. Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was measured under basal conditions and in the presence of dopamine, adenosine and prostaglandin (PG) E1. A clear dissociation occurred between the respective evolution patterns of basal and agonist-stimulated adenylate cyclase activities. The magnitudes of the enzyme response to PGE1, adenosine, and dopamine also exhibited different evolution patterns during the cell cycle. Evolution of adenylate cyclase responsiveness to PGE1 during the cell cycle exhibited striking similarities with the intracellular 3':5'-cyclic AMP changes observed elsewhere. Use of theophylline and fluphenazine as specific inhibitors of adenosine and dopamine, respectively, made it possible to demonstrate that adenosine, dopamine, and PGE1 stimulated adenylate cyclase through independent receptor sites. Furthermore, whatever the stage of the cell cycle, responses to these three agonists were not additive, indicating that the receptors of adenosine, dopamine, and PGE1 control the same adenylate cyclase moieties. The data suggest that adenylate cyclase cell content and enzyme responsiveness to specific agonists can be independently controlled.     

Lateral diffusion of lipids in membranes by pulse saturation recovery electron spin resonance.

Short-pulse saturation recovery electron spin resonance methods have been used to measure lateral diffusion of nitroxide-labeled lipids in multilamellar liposomal dispersions. Nitroxides with 14N and 15N isotopes introduced both separately and together were used. Differential equations have been written and solved for complex saturation recovery signals involving several superimposed exponentials. The time constants contain various combinations of the spin-lattice relaxation time (T1e) for both isotopes, Heisenberg exchange rates, and nuclear spin-lattice relaxation times (T1n). Signals of high quality were fitted by Monte Carlo variation of the amplitudes and time constants. The reliability of the approach was tested extensively by verifying that (i) the predicted number of exponentials agreed with the experimental number, (ii) relaxation parameters that were determined were independent of the observed hyperfine transition, (iii) the time constants were independent of saturating pulse length, (iv) T1e and T1n do not change when Heisenberg exchange is changed by varying the concentration, and (v) Heisenberg exchange is indeed proportional to the concentration. It has been established that bimolecular collision rates over a wide range of conditions can be reliably measured using the methodology described here. The methods depend on the favorable match of bimolecular collision rates at micromolar concentrations to nitroxide spin-lattice relaxation probabilities.     

Changes in microtubule phosphorylation during cell cycle of HeLa cells.

The phosphorylation in vitro and in vivo of tubulin isolated from HeLa cells has been examined during the cell cylce. The results obtained indicate that: (a) the protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity present in the microtubules, and measured in vitro with exogenous casein as substrate, is maximal in M cells, whereas that present in the cytosol is nearly constant during the S, G-2, and M stages, and decreases during G-1; (b) the patterns through the cell cycle of the maximal number of tubulin sites phosphorylated in vitro and of the microtubular protein kinase activity are similar; (c) the degree of tubulin phosphorylation in vivo is 2- to 3-fold higher in the microtubules isolated from the S and M stages of the cell cycle, than those from G-1 and G-2. This variable phosphate content of tubulin through the cell cycle suggests that such covalent modification might be important to enable tubulin to carry over some of its functions during the cell cycle.     

Deuterium magnetic resonance studies of the interaction of lipids with membrane proteins.

The deuterium magnetic resonance spectra of lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1-(16,16,16-trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The less-ordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2- to 3-fold decrease in relative amount from 2 to 22 degrees. The rate of exchange of lipid between the free and restricted environments is slower than 10(3)/sec. The significance of these phenomena is discussed.     

Selective osmotic effect on diffusion of plasma membrane lipids in maize protoplasts.

Osmotic levels in the range typically used during plant protoplast isolation and incubation were investigated with regard to effects on the lateral diffusion of lipid probes in the plasma membrane. The lateral diffusion coefficient of a fluorescent sterol probe in the plasma membrane of maize (Zea mays L.) root protoplasts in a medium containing 0.45 M mannitol was 4 times faster than when the medium contained 0.9 M mannitol. The lateral diffusion coefficient of a fluorescent phospholipid probe, however, did not change over this range of mannitol concentrations. Similar diffusion characteristics were observed when the medium contained trehalose instead of mannitol. Slower lateral diffusion of the sterol probe at higher osmolality was also observed when KCl/CaCl2-based osmotic media were used with protoplasts isolated by a mechanical, rather than by an enzymic, method. Extraction and quantitation of total lipids from protoplasts showed that both the phospholipid and sterol contents per protoplast decreased with increasing osmolality, while the sterol/phospholipid ratio increased. These results demonstrate that osmotic stress induces selective changes in both the composition and biophysical properties of plant membranes.     

Lateral translational diffusion of cytochrome c oxidase in the mitochondrial energy-transducing membrane

The degree of freedom for lateral translational diffusion by cytochrome c oxidase and other integral proteins in the energy-transducing membrane of the mitochondrion was determined by combining the use of an immunoglobulin probe monospecific for the oxidase with thermotropic lipid phase transitions. Lateral mobility of the oxidase was monitored by observing the distribution of the immunoglobulin probe on the membrane surface by deep-etch electron microscopy and by observing the distribution of intramembrane particles (integral proteins) in the hydrophobic interior of the membrane by freeze-fracture electron microscopy. Incubation of the membrane with the immunoglobulin resulted in a time-dependent clustering of predominantly large intramembrane particles. Low temperature-induced lipid phase transitions resulted in the close packing of all intramembrane particles and cytochrome c oxidase by lateral exclusion from domains of gel-state bilayer lipid and was completely reversible. However, when cytochrome c oxidase was crosslinked through an immunoglobulin lattice prior to returning the membrane to above the lipid phase transition temperature, small intramembrane particles rerandomized while the large oxidase-related particles remained clustered. These observations reveal that cytochrome c oxidase can diffuse laterally in the energy-transducing membrane, either independently of all other integral proteins or in physical union with one or more other integral proteins. In addition, many other as yet unidentified smaller integral proteins can diffuse independently of the oxidase.     

Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, "flat" cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16(INK4A), responds to RA with elevation of p18(INK4C), marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21(Cip1). These changes result in sustained cdk activity. Accordingly, overexpression of p21(Cip1) but not p16(INK4A) induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.     

Phosphorylation of lens fiber cell membrane proteins.

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.     

Changes in the composition of plasma membrane proteins during differentiation of embryonic chick erythroid cell.

Erythroid cells which are homogeneous with regard to stage of maturation are naturally available from the circulation of chick embryos at various times of development. This provides a convenient system for examining the changes in plasma membrane protein composition during red cell maturation. Plasma membranes are isolated from chick embryonic erythroid cells at various stages of maturation. Extensive characterization of the isolated membranes show that they are pure and their proteins undegraded. Analyses by sodium dodecyl sulfate/polyacrylamide gel electrophoresis show that both qualitative and quantitative changes occur in membrane protein composition during the early stage of erythroid differentiation. Specific proteins of red cell membrane such as "spectrin" and band three proteins are present in low levels in early erythroblasts but increase in their relative amounts with maturation. A steady-state membrane protein composition seems to be established by the late polychromatophilic erythroblast stage.     

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains

Fluorescent lipid and phospholipid probes were incorporated at 4°C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18°C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 μg/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of ≈3 × 10^-9 cm²/sec. After labeling at higher probe concentrations (≥100 μg/ml), two diffusing species were observed, with diffusion coefficients of ≈3 × 10^-9 cm²/sec (“fast”) and ≈5 × 10^-10 cm²/sec (“slow”). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 37°C (D = 1.1 × 10^-8 cm²/sec); (ii) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 18°C in the presence of 2 mM EGTA (D = 4.2 × 10^-9 cm²/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18°C or 37°C (D = 2.5 × 10^-8 cm²/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.     

Phase changes in membrane lipids in sickle red cell shed-vesicles and sickle red cells

Lipid phase transformations may occur in the membranes of sickle red cell shed-vesicles and sickle red cells. The presence of such phase changes could be important in sickle cell disease since membrane phase changes appear to contribute to the generation of antiphospholipid antibodies that are thrombophilic and occur in sickle cell disease. In the present study, we have evaluated sickle red cell shed-vesicles and sickle red cells for the presence of non-bilayer lipid phases using ³¹P-NMR spectroscopy. Results show that the spectra of both the shed-vesicles and the sickle red cells are compatible with the occurrence of non-bilayer phases in the membrane bilayers. The findings support the concept that these membranes could contribute to the generation of antiphospholipid antibodies in sickle cell disease. Am. J. Hematol. 58:177–182, 1998. © 1998 Wiley-Liss, Inc.     

Animal cell lysosomes rapidly exchange membrane proteins.

The lysosome has been chosen as a model to study the exchange of native membrane proteins within an organelle population. Heterologous lysosomes were brought together by vesicular stomatitis virus-mediated cell fusion. The distribution of lysosomal membrane protein was visualized by indirect immunofluorescence using species-specific monoclonal antibody. LAMP-2, a mouse lysosomal membrane protein, and HLAMP-B, a human lysosomal membrane protein, were found to transfer to Chinese hamster ovary cell sucrosomes (sucrose-swollen lysosomes). This transfer occurred in the presence of cycloheximide. The exchange of LAMP-2 and LIMP I, a rat lysosomal membrane protein, was observed between native lysosomes in a mouse (3T3)-rat (normal rat kidney) cell fusion. Extensive transfer/exchange was observed within 1.5-2 hr postfusion, which is consistent with the kinetics of endocytic content exchange between lysosomes. Both membrane protein and content transfer between lysosomes were inhibited by nocodazole, a disrupter of microtubules, as was endocytic delivery to sucrose-swollen lysosomes. In the presence of nocodazole, tubular lysosomes disappeared. Both tubular lysosomes and microtubules may be important for the transfer/exchange. The interspecies cell fusion/monoclonal antibody approach developed here should be readily applicable to determining if membrane protein exchange is a property of other organelles such as Golgi apparatus and mitochondria.     

Differential phosphorylation of nuclear nonhistone high mobility group proteins HMG 14 and HMG 17 during the cell cycle.

The phosphorylation of the high-mobility group (HMG) proteins at different stages of the cell cycle was studied in synchronized HeLa cells. HMG proteins were extracted and analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Although the molecular weight distribution of HMGs remains unchanged, their total amounts increase by as much as 20-25% in the G1 and S phases when compared with amounts in G2. However, the most significant finding is that there is a 7-fold increase of 32P incorporation into HMG 14 in the G2 phase compared with that in G1, and a 2-fold increase of 32P incorporation into HMG 17 in early S phase relative to the incorporation in the G1 and G2 stages. In contrast, HMG 1 and HMG 2 are not phosphorylated. The clear demonstration of differential phosphorylation of HMG 14 and 17 at specific stages of the cell cycle warrants a serious consideration of their role in tissue-specific maintenance of the altered chromatin structure characteristic of potentially active or actively transcribed chromatin domains.