Caveolin-1促进乳癌Hs578T耐药细胞株侵袭和转移

通过基因转染技术培育caveolin-1过表达乳癌HS578T耐药细胞株Hs578T/Dox cav-1和空载体细胞株Hs578T/Dox vector,探讨caveolin-1蛋白对耐药肿瘤细胞体内和体外侵袭转移能力的影响.由于caveolin-1表达的增加,Hs578T/Dox cav-1细胞形态变为长梭形,伪足更长而伸展,其粘附能力高于空载体Hs578T/Dox vector细胞株((0.897±0.163)vs(0.633±0.053),P<0.01).侵袭小室试验发现,Hs578T/Dox cav-1侵袭转移能力增强,培养6h迁徙进入微孔膜的细胞数比Hs578T/Dox vector细胞株明显增多((107.8±10.9)vs(80.8±8.07),P<0.01),侵袭破坏matrigel基质蛋白胶进入膜内的细胞数也明显增多((68.8±9.88)vs(25.6±5.41),P<0.01).悬浮培养的Hs578T/Dox cav-1细胞比Hs578T/Dox vector细胞更容易聚集,形成相对较致密的细胞团块,24h检测流式细胞凋亡指数下降((8.79±1.54)%vs(16.42±1.42)%,P<0.01).这表明其具有更强的抗失巢凋亡和继续生存的能力,为循环转移的肿瘤细胞定植前取了更多时间.Hs578T/Dox vector细胞在裸鼠皮下种植成瘤试验中不能成瘤,而Hs578T/Dox cav-1细胞接种15只裸鼠,全部形成肿瘤,平均直径(0.8±0.45)cm.发现一只成瘤裸鼠双肺可见瘤样肿块转移.HE病理组织染色见肿瘤细胞弥漫性分布,细胞核异型性明显.上述结果表明,caveolin-1对Hs578T耐药肿瘤细胞侵袭、转移和成瘤性具有明显的促进作用.     

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Influence of membrane fluidity on human immunodeficiency virus type 1 entry

For penetration of human immunodeficiency virus type 1 (HIV-1), formation of fusion-pores might be required for accumulating critical numbers of fusion-activated gp41, followed by multiple-site binding of gp120 with receptors, with the help of fluidization of the plasma membrane and viral envelope. Correlation between HIV-1 infectivity and fluidity was observed by treatment of fluidity-modulators, indicating that infectivity was dependent on fluidity. A 5% decrease in fluidity suppressed the HIV-1 infectivity by 56%. Contrarily, a 5% increase in fluidity augmented the infectivity by 2.4-fold. An increased temperature of 40 degrees C or treatment of 0.2% xylocaine after viral adsorption at room temperature enhanced the infectivity by 2.6- and 1.5-fold, respectively. These were inhibited by anti-CXCR4 peptide, implying that multiple-site binding was accelerated at 40 degrees C or by xylocaine. Thus, fluidity of both the plasma membrane and viral envelope was required to form the fusion-pore and to complete the entry of HIV-1.     

ABCA1 modulates the oligomerization and Golgi exit of caveolin-1 during HDL-mediated cholesterol efflux in aortic endothelial cells

Previously, the authors have shown that the molecular interaction between caveolin-1 and ATP-binding cassette transporter A1 (ABCA1) is associated with the high-density lipoprotein (HDL)-mediated cholesterol efflux pathway in aortic endothelial cells (ECs). This study analyzed the role ABCA1 plays in caveolin-1-mediated cholesterol efflux in aortic ECs. Knockdown of ABCA1 by siRNA in primary rat aortic ECs after cholesterol treatment did not affect caveolin-1 expression but led to the retention of caveolin-1 in the Golgi apparatus, impaired caveolin-1 oligomerization, and reduced cholesterol efflux. Immunoblotting assay and immunofluorescence microscopy demonstrated that HDL transiently up-regulated ABCA1 expression, induced caveolin-1 oligomerization, and promoted its Golgi exit, thereby enhancing cholesterol efflux. These HDL-induced events, however, were inhibited by down-regulation of ABCA1. It is concluded that HDL up-regulates ABCA1 expression, which in turn modulates the oligomerization and Golgi exit of caveolin-1 to enhance cholesterol efflux in aortic ECs.     

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.     

Acyl-CoA: 1-acyl-glycero-3-phosphoethanolamine O-acyltransferase and liver plasma membrane fluidity

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.     

Rapid cold-hardening increases membrane fluidity and cold tolerance of insect cells

The rapid cold-hardening (RCH) response not only confers dramatic protection against cold-shock (non-freezing) injury, but also "instantaneously" enhances organismal performance. Since cold-shock injury is associated with damage to the cell membrane, we investigated the relationship between RCH and changes in cold tolerance and membrane fluidity at the cellular level. None of the adult flies (Sarcophaga bullata) in the cold-shocked treatment group survived direct transfer to -8 degrees C for 2 h; in contrast, 64.5% of flies in the RCH group survived exposure to -8 degrees C. Differences between the treatment groups also were reflected at the cellular level; only 21.3% of fat body cells in the cold-shocked group survived compared to 68.5% in the RCH group. Using 31P solid-state NMR spectroscopy, we determined that membrane fluidity increased concurrently with rapid cold-hardening of fat body cells. This result suggests that membrane characteristics may be modified very rapidly to protect cells against cold-shock injury.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Display of membrane proteins on the heterologous caveolae carved by caveolin-1 in the Escherichia coli cytoplasm

Caveolae are membrane-budding structures that exist in many vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicles. Recently, it was shown that caveolae-like structures were formed in Escherichia coli through the expression of caveolin-1. This interesting structure seems to be versatile for a variety of biotechnological applications. Targeting of heterologous proteins in the caveolae-like structure should be the first question to be addressed for this purpose. Here we show that membrane proteins co-expressed with caveolin-1 are embedded into the heterologous caveolae (h-caveolae), the cavaolae-like structures formed inside the cell. Two transmembrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, Syntaxin 1a and vesicle-associated membrane protein 2 (VAMP2), were displayed on the h-caveolae surface. The size of the h-caveolae harboring the transmembrane proteins was ∼100 nm in diameter. The proteins were functional and faced outward on the h-caveolae. Multi-spanning transmembrane proteins FtsH and FeoB could be included in the h-caveolae, too. Furthermore, the recombinant E. coli cells were shown to endocytose substrate supplemented in the medium. These results provide a basis for exploiting the h-caveolae formed inside E. coli cells for future biotechnological applications.     

Caveolin-1 deficiency alters plasma lipid and lipoprotein profiles in mice

Caveolae are specialized membrane microdomains formed as the result of local accumulation of cholesterol, glycosphingolipids, and the structural protein caveolin-1 (Cav-1). To further elucidate the role of Cav-1 in lipid homeostasis in-vivo, we analyzed fasting and post-prandial plasma from Cav-1 deficient mice on low or on high fat diet. In total plasma analysis, an increase in ceramide and hexosylceramide was observed. In cholesteryl ester (CE), we found an increased saturated + monounsaturated/polyunsaturated fatty acid ratio in fasting plasma of low fat fed Cav-1(−/−) mice with increased proportions of CE16:1, CE18:1, CE20:3, and decreased proportions of CE18:2 and CE22:6. Under high fat diet HDL-CE, free cholesterol and pre-β-HDL were increased accompanied by a shift from slow to fast migrating α-HDL and expansion of apoE containing HDL. Our results demonstrate a significant role of Cav-1 in HDL-cholesterol metabolism and may reflect a variety of Cav-1 functions including modulation of ACAT activity and SR-BI function.     

Azurin interaction with the lipid raft components ganglioside GM-1 and caveolin-1 increases membrane fluidity and sensitivity to anti-cancer drugs

Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts’ markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.     

Modulation of membrane fluidity by catalytic hydrogenation affects the chilling susceptibility of the blue-green alga, Anacystis nidulans

All the changes, i.e. the phase separation temperature of thylakoid lipids, shift in the chilling induced increase of K⁺ permeability and decline in photosynthetic O₂-production, respectively, brought about by temperature acclimation in Anacystis nidulans, can be accomplished by homogeneous catalytic hydrogenation of the fatty acids, as well, using a new water-soluble Pd(II) complex, hitherto unknown in biological applications. Since the thermo-adaptation replaced by proper hydrogenation conducted under isothermal condition results in a similar modification of chilling susceptibility, it afforts direct evidence that chilling response is mediated by changing the degree of fatty acid unsaturation in Anacystis nidulans.     

Resolution of plasma membrane lipid fluidity in intact cells labelled with diphenylhexatriene

The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of $\text{N-}\text{bixinoyl}$ glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward.