Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape.

A fundamental feature of cellular plasma membranes (PMs) is an asymmetric lipid distribution between the bilayer leaflets. However, neither the detailed, comprehensive compositions of individual PM leaflets nor how these contribute to structural membrane asymmetries have been defined. We report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Phospholipid unsaturation is dramatically asymmetric, with the cytoplasmic leaflet being approximately twofold more unsaturated than the exoplasmic leaflet. Atomistic simulations and spectroscopy of leaflet-selective fluorescent probes reveal that the outer PM leaflet is more packed and less diffusive than the inner leaflet, with this biophysical asymmetry maintained in the endocytic system. The structural asymmetry of the PM is reflected in the asymmetric structures of protein transmembrane domains. These structural asymmetries are conserved throughout Eukaryota, suggesting fundamental cellular design principles.

Author Correction: Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Isolation of giant plasma membrane vesicles for evaluation of plasma membrane structure and protein partitioning.

Although investigation into the structure of eukaryotic cell membranes has been an intense focus of cell biology for the past two decades, definitive insights have been limited by the lack of coherent methods for the isolation of specific organelle membranes and the identification of membrane subdomains. Here we describe a method for the isolation of mammalian cell plasma membranes as Giant Plasma Membrane Vesicles (GPMVs) and strategies for imaging membrane lateral structure and quantification of protein partitioning between coexisting domains by fluorescence microscopy.

A simple indentation device for measuring micrometer-scale tissue stiffness.

Mechanical properties of cells and extracellular matrices are critical determinants of function in contexts including oncogenic transformation, neuronal synapse formation, hepatic fibrosis and stem cell differentiation. The size and heterogeneity of biological specimens and the importance of measuring their mechanical properties under conditions that resemble their environments in vivo present a challenge for quantitative measurement. Centimeter-scale tissue samples can be measured by commercial instruments, whereas properties at the subcellular (nm) scale are accessible by atomic force microscopy, optical trapping, or magnetic bead microrheometry; however many tissues are heterogeneous on a length scale between micrometers and millimeters which is not accessible to most current instrumentation. The device described here combines two commercially available technologies, a micronewton resolution force probe and a micromanipulator for probing soft biological samples at sub-millimeter spatial resolution. Several applications of the device are described. These include the first measurement of the stiffness of an intact, isolated mouse glomerulus, quantification of the inner wall stiffness of healthy and diseased mouse aortas, and evaluation of the lateral heterogeneity in the stiffness of mouse mammary glands and rat livers with correlation of this heterogeneity with malignant or fibrotic pathology as evaluated by histology.

Electrostatic contribution to the surface pressure of charged monolayers containing polyphosphoinositides.

Structural and functional studies of lateral heterogeneity in biological membranes have underlined the importance of membrane organization in biological function. Most inquiries have focused on steric determinants of membrane organization, such as headgroup size and acyl-chain saturation. This manuscript reports a combination of theory and experiment that shows significant electrostatic contributions to surface pressures in monolayers of phospholipids where the charge spacing is smaller than the Bjerrum length. For molecules with steric cross sections typical of phospholipids in the cell membrane (approximately 50 A(2)), only polyphosphoinositides achieve this threshold. The most abundant such lipid is phosphatidylinositol bisphosphate, which has between three and four charged groups at physiological conditions. Theory and experiment show that surface pressure increases linearly with phosphatidylinositol bisphosphate net charge and reveal crossing of high and low ionic strength pressure-area isotherms, due to opposing effects of ionic strength in compressed and expanded monolayers. Theory and experiment show that electrostatic effects are negligible for monolayers of univalent lipids, emphasizing the unique importance of electrostatic effects for lateral organization of polyphosphoinositides. Quantitative differences between theory and experiment suggest that attractive interactions between polyphosphoinositides, possibly mediated by hydrogen bonding, can lessen the effect of electrostatic repulsions.

HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties.

In addition to forming the selective filtration barrier for the renal glomerulus, podocytes maintain glomerular capillary architecture by opposing distending hemodynamic forces. To understand the relationship of cytoskeletal properties and the mechanical characteristics of podocytes, we studied filamin expression and distribution and measured cell membrane deformability in conditionally immortalized wild-type (WT) mouse podocytes, and in podocytes derived from a mouse model of HIV-associated nephropathy (HIVAN). In the WT cells, filamin and F-actin were localized at the periphery and in prominent stress fibers. In the HIVAN cells, filamin expression was reduced, and stress fibers were sparse. In a microaspiration assay, HIVAN cells ruptured under minimal negative pressure. Atomic force microscopy demonstrated that the WT cells had a stiffness of 17 kPa, whereas the value for the HIVAN cells was 4 kPa. These results demonstrate that the mechanical properties of WT and HIVAN podocytes are markedly different in a manner that is consistent with differences in the composition and arrangement of their cytoskeletons. The mechanical properties of the WT podocytes suggest that these cells can better maintain capillary integrity than the HIVAN podocytes and implicate pathological assembly of the cytoskeleton as a mechanism of HIVAN.