Quisqualate-Stimulated Phosphatidylinositol Breakdown in Rat Cerebellar Membranes

The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose-dependent manner in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Half-maximal activation of the quisqualate response required 0.15 microM GTP gamma S and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositides 4,5-bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as L-glutamate, trans-D,L-1-aminocyclopentyl-1,3-dicarboxylic acid, and ibotenate, but not N-methyl-D-aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide-specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.     

Electrical Breakdown of Bimolecular Lipid Membranes as an Electromechanical Instability

The bimolecular lipid membrane (BLM) is modeled as a bulk elastic layer subject to a compressive electric force caused by applied voltages. Analysis of this model shows that a compressive instability develops when the electric stress exceeds a critical value. This instability tends to crush the film and thus rupture it. The predicted breakdown voltage, when compared with measured values for phosphatidylcholine and cholesterol, shows fair agreement, considering the uncertainty in the estimate of elastic parameters.     

The Physics of Cell Membranes

An overview is given of the fundamental physics underlying the self-assembly, molecular organisation and electrical properties of the membranes that envelop living cells. These ultra thin (∼ 6 nm) membranes act as a diffusion barrier between the cell interior (cytoplasm) and the external medium. They consist basically of a bi-molecular film of lipid molecules in which are embedded functional proteins that perform a variety of functions, including energy transduction, signalling, transport of ions (and othermolecules), etc. Some examples are also presented of the fascinating and socially and commercially important applications of membrane biophysics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Actinide Transport Across Cell Membranes

Protactinium uptake into the normal liver does not exceed 3%, but when the phospholipid levels in the liver are elevated by administration of thioacetamide this uptake increases to 31%. Phosphatidic acid, which is absent from the normal liver, has been shown to extract protactinium into organic solvents. However, phosphatidylserine, a component of normal liver cell membranes, does not extract protactinium. It might be conjectured that this is why so little protactinium is taken up by the normal liver. The hypothesis is advanced that phosphatidylserine, which is known to complex plutonium, americium and curium, may regulate the uptake of these elements by liver.     

The Membranes of a Eukaryotic Cell

The evolution of internal membrane systems has introduced manyadditional control steps into pathways that, although basicallysimilar in prokaryotic cells, are less sophisticated and leavemany aspects up to chance. Temporal and spatial control of secretion,quality control of the proteins secreted or inserted into theplasma membrane, and exquisite control over the selective degradationof macromolecules, for example, are apparently indispensiblerequirements in multicellular organisms, but are relativelyunimportant in bacteria. Although complicated in detail, mostmembrane traffic in the eukaryotic cell can be reduced to afew basic principles. Such a reductionist's view provides aconceptual framework that allows the reader to organize an otherwiseoverwhelming amount of data on cellular membrane architectureand dynamics.     

Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.     

Enzyme Activities in Polarized Cell Membranes

The theoretical pH dependence of enzyme activities in membranes of low dielectric constant is estimated. It is shown that in biological membranes some types of enzymes may attain a limiting pH sensitivity such that an increment of only 0.2 pH unit (sufficient to induce action potentials in squid axons) causes a relative activity change of over 25%. The transients of enzyme activity generated by membrane depolarization and by pH increments in the bathing solution are discussed in relation to the transients of nervous excitation.     

Adaptation of Yeast Cell Membranes to Ethanol

A highly ethanol-tolerant Saccharomyces wine strain is able, after growth in the presence of ethanol, to efficiently improve the ethanol tolerance of its membrane. A less-tolerant Saccharomyces laboratory strain, however, is unable to adapt its membrane to ethanol. Furthermore, after growth in the presence of ethanol, the membrane of the latter strain becomes increasingly sensitive, although this is a reversible process. Reversion to a higher tolerance occurs only after the addition of an energy source and does not take place in the presence of cycloheximide.     

Measurement of Dipole Potential in Bilayer Lipid Membranes by Dielectric Spectroscopy

Planar bilayer lipid membranes formed from egg phosphatidylcholine in aqueous media containing the lipophilic anion, dipicrylamine (DPA), were studied by dielectric spectroscopy over a frequency range of 10 Hz–10 MHz. The membranes showed dielectric relaxation due to the translocation of DPA between the membrane interfaces. Incorporating either cholesterol or 6-ketocholestanol into the membranes increased the characteristic frequency of the relaxation, which is proportional to the translocation rate constant of DPA. The results suggested that the sterol dipoles induced positive potential changes within the membrane interior. The changes of the dipole potential were 70 mV for cholesterol and 150 mV for 6-ketocholestanol when the sterol mole fraction was 0.67. The opposite effect was caused by phloretin added to the aqueous media, and the maximum dipole potential change was ?90 mV at 100 μM.     

Active Calcium Transport by Plant Cell Membranes

The cytosolic free calcium concentration of higher plant cellsis maintained at about 01 µM by the action of membranecalcium transporters. These act to remove calcium from the cytosoland expel it to the apoplast or accumulate it in intracellularstores. In this review, the properties and subcellular localizationsof these systems are described. The major calcium transporterof the plasma membrane is a calcium pumping ATPase which showsmany similarities to its equivalent in mammalian cells. Thetransporter has been purified from maize coleoptiles and isof M_r 140 000, binds (and is activated by) calmodulin and showscommon antigenicity with the mammalian protein. Higher plantendoplasmic reticulum also contains a calcium pumping ATPasewhich transports calcium from the cytoplasm and its role andproperties, together with those of the tonoplast calcium/protonantiporter are presented. Evidence for calcium accumulationby chloroplasts and mitochondria is considered. The review alsodeals with the regulation of plant cell membrane calcium transportand its role in providing intracellular pools of calcium forsignal transduction. Key words: Plant, calcium transport, ATPase, cell membrane, calmodulin     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

A Parallel Study of Pigment Bleaching and Cytochrome Breakdown during Aging of Thylakoid Membranes

Aging of freshly isolated thylakoid membranes from spinach leaves(Spinacia oleracea L.) leads to dramatic alterations in boththe cytochrome (b_{559 (HP)} and f) composition and pigment (chlorophyllsa and b and ß-carotene) content. These changes occurat a faster rate under anaerobic conditions or after heatingthylakoid membranes, and in light as well as in darkness. Inaddition, when thylakoid membranes are heated at 78°C for8 min, or incubated in the presence of an emulsion of linoleicacid, a huge decrease in both cytochrome (particularly cyt.b_{559 (HP)}) and pigment contents occur. Whatever the experimentalconditions, cytochrome b_{559 (HP)} destruction occurs as soonthe aging process starts. Conversely, pigment bleaching is detectableafter an initial lag phase of about 60–70 min. Then, thetwo processes (cytochrome breakdown and bleaching of pigments)appear to take place in parallel. The addition of salicylhydroxamicacid or 8-hydroxy-quinoline, two radical scavenger components,to the aging medium strongly reduces the rate and extent ofcytochrome breakdown and pigment bleaching. On the basis of these results, a tentative scheme accountingfor the bleaching of pigments and the breakdown of cytochromesduring aging in vitro of thylakoid membranes is proposed. Itis suggested that these changes are mediated via a non-enzymaticmechanism in which free radicals could be implicated. The possiblerole of free radicals inducing ultrastructural changes at thelevel of chloroplast membranes in senescent leaves is also considered. (Received October 11, 1985; Accepted January 24, 1986)     

Penetration of Host Cell Membranes by Adenovirus 2

Highly purified human adenovirus type 2 directly penetrated the plasma membranes of KB cells. The process of membrane penetration resulted in the appearance of large numbers of adenovirions free in the cytoplasm of the infected cells. The virions underwent a morphological change as they penetrated the cell surface. Penetration of the plasma membranes and the accompanying alteration in virion morphology was dependent on a function associated with the intact cells, because neither event occurred when purified virions were added to isolated cell membranes. Inactivation of the adenovirions with heat or antibodies before inoculation of the cells reduced the infectivity of the virus population and prevented the appearance of virions free in the cytoplasm. The inactivation of the virions did not significantly reduce the number of virus particles which were found in cell vacuoles and pinocytotic vesicles.     

Strain Energy Function of Red Blood Cell Membranes

The several widely different values of the elastic modulus of the human red blood cell membrane which have been reported in the literature are incorporated into a single strain energy function consisting of two terms. One term gives the small stresses and low elastic modulus which is observed when the red cell membrane is deformed at constant area. The second term contributes a large isotropic stress dependent on the change of area. The strain energy function is applied to the process of sphering of red blood cells in a hypotonic solution. It is shown that a nearly perfect sphere can result even though the red blood cell membrane is homogeneous in all areas of the cell. Results pertinent to sieving and micropipette experiments are also explored.     

Phase-Separated Structures of Sake Flavors-Containing Cell Model Membranes

Sake (a traditional Japanese alcoholic beverage) contains ethyl caproate (EC), which enhances its economic value. Isovaleraldehyde (IVA) is also a well-known flavoring agent in alcoholic beverages, which some people enjoy. Recently, studies revealed that EC decreased the size of homogenous 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes whereas IVA increased their size. Cholesterol (Chol) and ergosterol were previously referred to as animal and fungus sterols. For the first time, this study demonstrated the phase behavior of the membrane in cell-sized liposomes containing EC and IVA. After adding EC, the solid ordered/liquid disordered (Ld) and liquid ordered (Lo)/Ld phase separation in DOPC/dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol or ergosterol ternary membranes decreased, but the Lo/Ld phase separation decreased after adding IVA. Biophysics, physiological, and application aspects of EC and IVA evaluation were discussed. The findings of this study not only enhance our understanding of the function of flavors but also provide rapid and cost effective performance for the measurement.     

ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes

The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME).

This is a PLoS Computational Biology Software Article.

     

Dielectric Relaxation Dynamics of Water in Model Membranes Probed by Terahertz Spectroscopy

We study hydrated model membranes, consisting of stacked bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids, using terahertz time-domain spectroscopy and infrared spectroscopy. Terahertz spectroscopy enables the investigation of water dynamics, owing to its sensitivity to dielectric relaxation processes associated with water reorientation. By controlling the number of water molecules per lipid molecule in the system, we elucidate how the interplay between the model membrane and water molecules results in different water dynamics. For decreasing hydration levels, we observe the appearance of new types of water dynamics: the collective bulklike dynamics become less pronounced, whereas an increased amount of both very slowly reorienting (i.e., irrotational) and very rapidly reorienting (i.e., fast) water molecules appear. Temperature-dependent measurements reveal the interconversion between the three distinct types of water present in the system.     

顺铂与小鼠艾氏腹水肝癌细胞膜的相互作用

本文研究了顺铂对小鼠艾氏腹水肝癌细胞膜蛋白内源性荧光的淬灭作用和测定了其在膜上的结合量。结果表明顺铂能与癌细胞膜结合。按存在两类结合部位,得到表观结合常数和结合部位数为: K_1=1.35×10~5L/mol n_1=6.80×10~(-4)mol/g(protein) K_2=2.50×10~3L/mol n_2=1.92×10~(-3)mol/g(protein)     

Exciting Cell Membranes with a Blustering Heat Shock

Brief heat shocks delivered to cells by pulsed laser light can evoke action potentials in neurons and contraction in cardiomyocytes, but the primary biophysical mechanism has been elusive. In this report we show in the neuromuscular junction of Caenorhabditis elegans that application of a 500°C/s heat shock for 500 μs evoked ∼35 pA of excitatory current and injected ∼23 fC(femtocoulomb) of charge into the cell while raising the temperature only 0.25°C. The key variable driving the current was the rate of change of temperature (dT/dt heat shock), not temperature itself. The photothermal heat shock current was voltage-dependent and was from thermally driven displacement of ions near the plasma membrane. The charge movement was rapid during the heat shock and slow during thermal relaxation, thus leading to an asymmetrical capacitive current that briefly depolarized the cell. A simple quantitative model is introduced to describe modulation of the membrane potential and facilitate practical application of optical heat shock stimuli.     

Molecular Engine for Transporting Drugs Across Cell Membranes

Abstract

A molecular engine has been developed from first principles to transport drugs from endosomes to the cytosol of cells. The engine is powered by the pH differential across the endosomal membrane, does not disrupt the endosomal membrane, and is disassembled into innocuous components after carrying out its transport function.