Theoretical approach to the description of microvesicular transport in the endothelium

Theoretical consideration of mechanisms on curvature of the cell membrane, resulting in formation of microvesicles in the cells has been carried out. These mechanisms are presented as: 1) local changes of membrane surface tension at the border membranes--external medium; 2) local changes of the membrane potential; 3) hydrostatic pressure; 4) crystallization of membrane domains; 5) enzymatic changes of the lipid molecule configurations. Energy of the curved membrane and the energy of the external effects for the vesicle are estimated. Spontaneous formation of vesicles at the expense of the oscillatory mechanism is impossible. The most probable mechanism, from the energetic point of view, for vesicular formation is adsorption of surface-active substances or ions on the membrane. Thus, the processes of microvesicular mass transfer, playing an important role in transcapillary mass exchange are determined by physicochemical mechanisms. From the physico-chemical point of view adsorption of substances can be the most probable mechanism in the vesicular formation and determine intensity of vesicular transport.     

Formation of the tectorial membrane of the cochlear canal during the embryogenesis of birds

By means of electron microscopy formation of the tectorial membrane of the cochlear canal and differentiation of the cells participating in the process (supporting cells of the basilar papilla and anterior homogeneous cells--AHC) have been studied in chick embryos. The AHC, to which the tectorial membrane is fixed, produce fine fibrillar material, included into the composition of the tectorial membrane. The cells mentioned form a number of cytoskeletal structures connected with the mechanical function of the tectorial membrane. Besides the network of the tonofilaments, gradually filling cytoplasm of the AHC, some peculiar attachings in the form of collagenous fibrillar bundles are revealed, they reach the AHC from the sublying connective tissue and have a direct contact with the basal membrane of the cells. The beginning of the tectorial membrane formation precedes the formation of the cytoskeletal structures. The latter appear only when the mass of the tectorial membrane, and hence, the mechanical loading on the AHC is great enough.     

Hepatocyte growth factor inhibits the formation of the basement membrane of alveolar epithelial cells in vitro

Hepatocyte growth factor (HGF) is a pulmotrophic factor for the regeneration of injured pulmonary tissue. We investigated the role of HGF in basement membrane formation during wound healing by immortalized alveolar type II epithelial cells that could form a continuous basement membrane when they were cultured on collagen fibrils in the presence of entactin-contaminated laminin-1. Cells cultured with 5.0 ng/ml HGF neither formed a continuous basement membrane on collagen fibrils nor maintained a continuous basement membrane architecture on a basement membrane substratum. The cells showed increased secretion of matrix metalloproteinase-9 and urokinase-type plasminogen activator, and the HGF-induced inhibition of basement membrane formation was attenuated by addition of 200 ng/ml tissue inhibitor of matrix metalloproteinase-1. Cells sequentially exposed to HGF and 1.0 ng/ml transforming growth factor-beta1 had enhanced basement membrane formation compared with those receiving these reagents in the reverse order or concurrently. HGF simultaneously stimulated proliferation and migration of the cells so that it advanced wound closure on the basement membrane substratum. The present results indicate that the role of HGF in wound healing is the stimulation of reepithelization, but this factor may also contribute to the degradation of the basement membrane.     

Cuticle formation in the embryo of Drosophila melanogaster

In Drosophila melanogaster embryos cuticle formation occurs between 12 and 16 hours of development at 25°C. The formation of the cuticulin and the protein epicuticular layers is simultaneous in the hypoderm, the tracheoblasts, and the fore- and hindgut cells. The cuticulin forms as a dual lamina, aggregating from granules secreted by the hypodermal cells. This is followed by the formation of a granular protein epicuticle and finally by the secretion of a mixed fibrous and granular endocuticle. All secretory cells are relatively simple in their ultrastructure. The secretory process is a membrane phenomenon, occurring at the tips of hypodermal microvillae on cells at the surface of the embryo and on those hypodermal cells lining the lumen of the fore- and hindgut. It also occurs along the entire surface of the tracheoblast lumen as well as on the outer surface of those cells which form exoskeletal chitinous setae. The process involves a specialization of the plasma membrane with the formation of secretory granules intracellularly beneath the membrane and the extrusion of these granules through the membrane to the outside where final cuticle formation occurs.     

Ksenia Guseva: Formation and Cooperative Behavior of Protein Complexes on the Cell Membrane

The thesis as a book on formation and cooperative behavior of protein complexes on the cell membrane highlights three major sections on protein oligomerization on cell surface, review approaches on membrane protein oligomerization and validation of oligomer formation process mainly using pure physical/theoretical models. These chapters cover an existing knowledge on membrane protein oligomer formation, experimental approaches with focus on mechanosensitive channels, interactions in oligomer assembly/stability, protein fragmentation, and pore formation in Tat complex system. In almost every chapter, physical/theoretical models have been integrated. A merger of protein oligomerization phenomena and theoretical physics is not quite intriguing for biologists/biochemists lacking adequate knowledge in physical modeling and its theoretical applications, and vice versa.     

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

Effect of detergents on the ATPase activity and barrier properties of the plasma membrane of smooth muscle cells

The effect of detergents on enzymic and barrier properties of membrane structures is studied in the plasma membrane fraction, postsynaptic membranes of smooth muscle cells and sterine bilipid membranes. The formation of hydrophylic pores in the membrane, as well as changes in the phase state of the lipid matrix and fluidity of lipid microenvironment of membrane enzymes are supposed.     

A novel technique for the study of cake formation during cross-flow microfiltration of microbial cells

Summary A novel technique involving the measurement of cell concentration changes in the suspension recycle reservoir was used to study cake formation in cross-flow microfiltration. Using this technique, cake formation was found to be enhanced by increasing transmembrane pressure and decreasing cross-flow velocity, and to be more pronounced on a microfiltration membrane than on an ultrafiltration membrane of equal hydraulic resistance. In the case of the microfiltration membrane, it was found that the build-up of cake was not a completely reversible process.     

Key phases in the formation of caveolae

Caveolae are abundant plasma membrane pits formed by the coordinated action of peripheral and integral membrane proteins and membrane lipids. Here, we discuss recent studies that are starting to provide a glimpse of how filamentous cavin proteins, membrane-embedded caveolin proteins, and specific plasma membrane lipids are brought together to make the unique caveola surface domain. Protein assembly involves multiple low-affinity interactions that are dependent on ‘fuzzy’ charge-dependent interactions mediated in part by disordered cavin and caveolin domains. We propose that cavins help generate a lipid domain conducive to full insertion of caveolin into the bilayer to promote caveola formation. The synergistic assembly of these dynamic protein complexes supports the formation of a metastable membrane domain that can be readily disassembled both in response to cellular stress and during endocytic trafficking. We present a mechanistic model for generation of caveolae based on these new insights.     

Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation

When cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube-like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (α_vβ₃) and fibronectin (α₅β₁) receptors inhibited cord formation. Subunit-specific monoclonal antibodies to α_v, β₃, and β₁ integrin subunits inhibited cord formation, while monoclonal antibodies to α₃ did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12-myristate 13-acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord-forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C-shaped cord-forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation. © 1993 Wiley-Liss, Inc.     

Immunocytochemical Localization of Enzymes Involved in Lignification of the Cell Wall

O -methyltransferase, and cinnnamyl alcohol dehydrogenase were localized to differentiating xylem. These enzymes are particularly abundant during secondary wall formation. Immunolabeling was observed on polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of anionic peroxidase was also localized to the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough endoplasmic reticulum (r-ER), the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized on the plasma membrane by fusion of the Golgi vesicles to the membrane. Received 3 September 2001/ Accepted in revised form 16 October 2001     

Mechanics of the Formation,Interaction, and Evolution of Membrane Tubular Structures

Membrane nanotubes, also known as membrane tethers, play important functional roles in many cellular processes, such as trafficking and signaling. Although considerable progresses have been made in understanding the physics regulating the mechanical behaviors of individual membrane nanotubes, relatively little is known about the formation of multiple membrane nanotubes due to the rapid occurring process involving strong cooperative effects and complex configurational transitions. By exerting a pair of external extraction upon two separate membrane regions, here, we combine molecular dynamics simulations and theoretical analysis to investigate how the membrane nanotube formation and pulling behaviors are regulated by the separation between the pulling forces and how the membrane protrusions interact with each other. As the force separation increases, different membrane configurations are observed, including an individual tubular protrusion, a relatively less deformed protrusion with two nanotubes on its top forming a V shape, a Y-shaped configuration through nanotube coalescence via a zipper-like mechanism, and two weakly interacting tubular protrusions. The energy profile as a function of the separation is determined. Moreover, the directional flow of lipid molecules accompanying the membrane shape transition is analyzed. Our results provide new, to our knowledge, insights at a molecular level into the interaction between membrane protrusions and help in understanding the formation and evolution of intra- and intercellular membrane tubular networks involved in numerous cell activities.     

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.     

Alzheimer's disease: its origin at the membrane, evidence and questions

Numerous results on membrane lipid composition from different regions of autopsied Alzheimer's disease brains in comparison with corresponding fractions isolated from control brains revealed significant differences in serine- and ethanolamine-containing glycerophospholipid as well as in glycosphingolipid content. Changes in membrane lipid composition are frequently accompanied by alterations in membrane fluidity, hydrophobic mismatch, lipid signaling pathways, transient formation and disappearance of lipid microdomains, changes in membrane permeability to cations and variations of other membrane properties. In this review we focus on possible implications of altered membrane composition on beta-amyloid precursor protein (APP) and on proteolysis of APP leading eventually to the formation of neurotoxic beta-amyloid (A beta) peptides, the major proteinaceous component of extracellular senile plaques, directly involved in Alzheimer's disease pathogenesis.     

Observations on the Formation and Ultrastructure of the Egg Membrane in the fern Ceratopteris thalictroides (Parkeriaceae)

The mature egg of ferns possesses a layer of egg membrane in the periphery. However, the fine trastructure and the formation of the egg membrane in the egg of ferns are still unclear. The present paper described the formation and the ultrastructure of the egg membrane in the egg of the fern Ceratopteris thalictroides using transmission electron microscopy (TEM). The results show that the egg membrane begins to form at the stage of the maturing egg. The egg membrane in the upper side of the egg is prominent. It is formed by attaching of osmiophilic sheets of endoplasmic reticulum to the inner surface of the plasmalemma . When mature the egg membrane in the upper side of the egg is much thick in the central region and it becomes thinner gradually towards the margin. Some osmiophilic materials are filled in the spaces between the sheets of endoplasmic reticulum . On the contrary, the egg membrane in the lower side of the egg is thin . It is composed of twoosmiophilic membranes which are associated with each other closely. The formation and the ultrastructure of the egg membrane are described for the first time . Some functions of the egg membrane are also discussed .     

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.     

Regulation of neuronal SNARE assembly by the membrane

In the neuron, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) assembly acts centrally in driving membrane fusion, a required process for neurotransmitter release. In the cytoplasm, vesicular SNARE VAMP-2 (vesicle-associated membrane protein-2) engages with two plasma membrane SNAREs, syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa), to form the core complex that bridges two membranes. Although various factors regulate SNARE assembly, the membrane also aids in regulation by trapping VAMP-2 in the membrane. Fluorescence and EPR analyses revealed that the insertion of seven C-terminal core-forming residues into the membrane controls complex formation of the entire core region, even though the preceding 54 core-forming residues are fully exposed and freely moving. When two interfacial tryptophan residues in this region were replaced with hydrophilic serine residues, the mutation supported rapid complex formation. The results suggest that the membrane-proximal region of VAMP-2 is a regulatory module for SNARE assembly, providing new insights into calcium-triggered membrane fusion.     

Condensation of the plasma membrane at the site of T lymphocyte activation

After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.     

Quantitative study of the importance of water permeability in plant cold hardiness

The rate of ice formation was measured for Hedera helix L. cv. Thorndale (English ivy) bark exposed to -10 C. The cooling rate of bark exposed to -10 C was 31 C per minute. The water efflux rate required for ice formation to occur extracellularly was calculated from the rate of ice formation and the average cell diameter. The water potential difference driving the efflux of water to sites of extracellular ice was calculated from the sample temperature, osmotic water potential, and fraction of water frozen at a given freezing temperature. From the water efflux rate and water potential difference, the resistance of the barrier controlling movement of intracellular water to sites of extracellular ice was calculated. Comparison of the resistance of this barrier to water movement with the resistance of the cell membrane revealed that the membrane represented only 0.5% of the barrier resistance. Thus, membrane resistance can have little influence on the rate of water efflux and ice formation when bark is cooled at a rate of 31 C per minute. If ice formation occurred at the same rate in ivy bark as it occurred in a 10 mm MnCl(2) solution, the membrane resistance would still have represented only 1% of the resistance of the barrier to ice formation. Therefore, at a cooling rate of 31 C/minute, heat removal plays a large part in determining the rate of ice formation. At slower cooling rates experienced under natural freezing conditions the ability to remove heat would play an even larger role. It is concluded that under natural freezing conditions membrane resistance does not limit water efflux.     

Effect of some amphiphilic drugs on the membrane morphology and aggregation of rabbit platelets

Treatment of normal, disc-shaped rabbit platelets with lysophosphatidylcholine and chlorpromazine induced respectively spine formation and spherical transformation. In similar concentration ranges to those in which they induced these morphological changes, the drugs suppressed a series of events triggered by thrombin: pseudopod formation, arachidonate release from the membrane phospholipids, and aggregation. Washing the drug-treated platelets reversed the morphological changes and abolished the inhibitory effect on aggregation. These observations suggest that amphiphilic drugs perturb the plasma membrane structure of platelets, inducing the membrane shape change and inhibiting the stimulus-induced aggregation.