Actual Cell Membranes Research Articles

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed.

Entropic force between membranes reexamined

Lipid bilayers serve as model surfaces that are often used to understand the behavior of actual cell membranes. Cell adhesion (1, 2), membrane fusion (1), binding-unbinding transition (3), self-assembly, and related phenomena govern an astounding array of biological functions; for example, sperm-egg fusion is the basis of mammalian reproducion (1). These phenomena are dictated by a complex interplay between the various attractive and repulsive forces that mediate between biological membranes. The key role is played by a repulsive force termed “steric hindrance,” or simply entropic pressure, the origins of which lie in the thermally excited fluctuations of membranes. Nearly four decades ago, in a landmark paper, Wolfgang Helfrich (4) proposed both the concept and the quantitative nature of this force. Freund (5) reexamines this paradigm in PNAS, and finds that the entropic force is of a remarkably different character than hitherto believed.

Imaging of Lipid Bilayer Mixtures and Actual Cell Membrane Fragments by Nanosims

Lipids, proteins, and cholesterol are the major components of biological membranes; however, little is known about their lateral organization and how it changes during cell-cell interactions, signaling, development and division on the nanometer length scale that is relevant for function. These studies develop a new method for determining the spatial composition and organization of model membranes using secondary ion mass spectrometry (SIMS) imaging in an effort to explain the rich phase behavior of complex biological membranes. Furthermore, these experiments set the stage for measuring the native distribution of cholesterol and proteins within membrane fragments taken from cells. Advanced fluorescence and atomic force microscopies (FM and AFM, respectively) used thus far to interrogate model cell membranes have limited molecular specificity (can only detect fluorescently labeled molecules in FM), spatial resolution (governed by optics in FM or tips in AFM) and both lack compositional information. SIMS imaging, in particular using the NanoSIMS by Cameca, offers imaging capability with high sensitivity (sub-ppm), specificity (based on isotopic labeling), and spatial resolution (∼50nm). Using this unique technique, the composition and organization of cholesterol, lipids and proteins within the plane of the lipid bilayer of model and actual cell membranes with nanometer resolution is investigated. Cholesterol in plasma membranes is responsible for modulating acyl chain order, membrane elasticity and lateral organization and its level is tightly regulated. In order to investigate the spatial distribution of cholesterol in actual cell membranes, supported cell membrane fragments are imaged using the NanoSIMS. These measurements are the first example of the direct analysis of the organization of a cell membrane at the nanometer length scale that is relevant for function setting the stage for quantitative analysis of systems of increasing complexity and direct biological relevance.

Transport of small molecules across cell membranes: water channels and urea transporters.

How do small hydrophilic nonelectrolytes cross cell membranes? Which pathways are most important for small lipid insoluble molecules to cross cell membranes? These are questions that have been basic to membrane transport physiology for decades. More importantly, these are questions whose answers have changed significantly within the last 10 years. This review discusses the evidence that pathways other than the lipid bilayer itself exist for the transport across cell membranes of specific small hydrophilic nonelectrolytes. The description begins with briefly analyzing the relevance of well accepted basic mathematical models for transport for understanding the permeability of representative physiologically important molecules across actual cell membranes. Particular emphasis is placed on describing recently discovered proteins that facilitate the transport of some of the smallest physiologically important lipid-insoluble molecules, water, and urea. Evidence also exists for transport proteins that selectively enhance the transmembrane transport of other small lipid-insoluble molecules. Do nonselective pores for small molecules exist in cell membranes?

Quantifying Cell−Surface Interactions Using Model Cells and Total Internal Reflection Microscopy

Nonspecific colloidal forces play an important role in controlling the adhesion of cells and liposomes to surfaces. A better understanding of cell−surface interactions is therefore needed for the development of improved drug delivery systems and biomemetic materials. The nonspecific interactions between model cells and a glass plate at low ionic strengths were quantified using total internal reflection microscopy (TIRM). Model cells were developed using 4.32 and 9.98 μm polystyrene spheres coated with phospholipids. Varying ratios of zwitterionic dipalmitoyl phosphatidylcholine (DPPC) and negatively charged dimyristoyl phosphatidylglycerol (DMPG) were used to coat the spheres to mimic the lipid charge composition of actual cell membranes. Measured potential energies of interaction between the model cells and a glass plate show excellent agreement with theoretical predictions based on an exponential model for the electrostatic energy. Debye lengths calculated from the experiments agree well with those predicted from solution conductivity measurements. The data presented show that TIRM is very effective in measuring interaction forces in model biological systems and can be used to screen potential stabilizing molecules for use in liposomes and biomimetic materials.

Hemagglutination and the closest distance of approach of normal, neuraminidase- and papain-treated erythrocytes.

By means of potential energy versus distance diagrams, derived from electrophoretic and surface tension data, the minimum distances of approach of normal (NOR) and of neuraminidase-treated (NEU) and papain-treated (PAP) human erythrocytes could be determined. The minimum distances between the actual cell membranes of two opposing red cells are: 184 A (NOR), 111 A (NEU), and 113 A (PAP), which agrees well with the fact that anti-D (Rho) antibodies of the IgG-class (which have a maximum distance of approximately 120 A between the two antibody-active sites) can hemagglutinate NEU and PAP cells, but are incapable of hemagglutinating normal D (Rho)-positive erythrocytes.

Zeta potentials, van der Waals forces and hemagglutination.

It has recently become possible to determine the van der Waals (Hamaker) coefficient of erythrocytes, whilst their zeta-potential has been known for some time. With these two data the net potential energy of interaction versus distance diagrams could be elaborated for unsensitized human erythrocytes suspended in saline water, as well as for erythrocytes monogamously sensitized with anti-D (Rh0) antibodies of the IgG class. Unsensitized erythrocytes can approach each other, to within approximately equal to 79 A of their sialoglycoprotein surfaces, leaving a distance between their actual cell membranes of approximately equal to 180 A, which is considerably more than the maximum distance between the two valencies of an IgG molecule (approximately equal to 120 A). This explains why unaided anti-D (Rh0) antibodies of the IgG class cannot cross-link two D (Rh0)-positive erythrocytes, although cross-linking can easily be achieved with IgM class antibodies. D (Rh0)-positive erythrocytes, monogamously sensitized with antibodies of the IgG class, can approach each other to within approximately equal to 60 A (between the Fc ends of the protruding antibodies), which makes cross-linking by means of anti-IgG antibodies of the IgG class feasible.