Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.

During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating proteinsorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrinbased skeleton. IntroductionDuring mammalian erythroid terminal differentiation, the plasma membrane and cytoskeleton are in a state of dynamic reorganization. We and others have determined that changes in protein expression and membrane protein assembly occur that involve both integral and skeletal membrane components. [1][2][3][4][5][6][7] At the conclusion of terminal differentiation, erythroblasts expel their nuclei and become reticulocytes. During enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is the sorting of erythroblast plasma membrane components to the plasma membranes of the nascent reticulocyte and the expelled nucleus. Although approximately 2 million reticulocytes are generated each second, amazingly little is known about molecular mechanisms governing protein sorting during enucleation. It is known that actin, spectrin, tubulin, ankyrin, and protein 4.1 partition to young reticulocytes, leaving extruded nuclei devoid of skeletal elements. 3,[8][9][10] Earlier studies also report that nonsialated glycoproteins are enriched in membranes of extruded nuclei, while sialoglycoproteins are enriched in membranes of young reticulocytes. 11 However, the redistribution of a large number of well-characterized integral membrane proteins and the mechanism(s) underlying their redistribution are unexplored.Since glycophorin A (GPA) is a well-defined, major sialoglycoprotein in the mature erythrocyte (as reviewed in Chasis and Mohandas 12 ), we chose it as a model integral membrane ...

Mitochondrial dysfunction has been implicated in the pathophysiology of Alzheimer's disease (AD) brains. To unravel the mechanism(s) underlying this dysfunction, we demonstrate that phospholipases A 2 (PLA 2 s), namely the cytosolic and the calcium-independent PLA 2 s (cPLA 2 and iPLA 2 ), are key enzymes mediating oligomeric amyloid-␤ peptide (A␤ 1-42 )-induced loss of mitochondrial membrane potential and increase in production of reactive oxygen species from mitochondria in astrocytes. Whereas the action of iPLA 2 is immediate, the action of cPLA 2 requires a lag time of ϳ12-15 min, probably the time needed for initiating signaling pathways for the phosphorylation and translocation of cPLA 2 to mitochondria. Western blot analysis indicated the ability of oligomeric A␤ 1-42 to increase phosphorylation of cPLA 2 in astrocytes through the NADPH oxidase and mitogen-activated protein kinase pathways. The involvement of PLA 2 in A␤ 1-42 -mediated perturbations of mitochondrial function provides new insights to the decline in mitochondrial function, leading to impairment in ATP production and increase in oxidative stress in AD brains.

To assess local elasticity in the red cell's spectrin-actin network, nano-particles were tethered to actin nodes and their constrained thermal motions were tracked. Cells were both immobilized and controllably deformed by aspiration into a micropipette. Since the network is well-appreciated as soft, thermal fluctuations even in an unstressed portion of network were expected to be many tens of nanometers based on simple equipartition ideas. Real-time particle tracking indeed reveals such root-mean-squared motions for 40-nm fluorescent beads either tethered to actin directly within a cell ghost or connected to actin from outside a cell via glycophorin. Moreover, the elastically constrained displacements are significant on the scale of the network's internodal distance of approximately 60-80 nm. Surprisingly, along the aspirated projection-where the network is axially extended by as much as twofold or more-fluctuations in the axial direction are increased by almost twofold relative to motions in the unstressed network. The molecular basis for such strain softening is discussed broadly in terms of force-driven transitions. Specific considerations are given to 1) protein dissociations that reduce network connectivity, and 2) unfolding kinetics of a localized few of the red cell's approximately 10(7) spectrin repeats.

Blood-brain barrier (BBB) dysfunctions have been implicated in the development and progression of Alzheimer's disease. Cerebral endothelial cells (CECs) and astrocytes are the main cell components of the BBB. Although amyloid-β oligomers (Aβ42) have been reported to mediate oxidative damage to the CECs and astrocytes and trigger the downstream MAPK/ERK pathway, the cell surface binding site for Aβ42 and exact sequence of these events have yet to be elucidated. In this study, the receptor for advanced glycation endproducts (RAGE) was postulated to function as a signal transducing cell surface receptor for Aβ42 to induce ROS generation from NADPH oxidase and trigger downstream pathways for the phosphorylation of extracellular-signal-regulated kinases (ERK1/2) and cytosolic phosphorilase A2 (cPLA2). We found that Aβ42 competed with the anti-RAGE antibody (AbRAGE) to bind to RAGE on the surfaces of CECs and primary astrocytes. In addition, AbRAGE abrogate Aβ42-induced ROS production and the co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase in both cell types. AbRAGE, as well as NADPH oxidase inhibitor and ROS scavenger suppressed Aβ42-induced ERK1/2 and cPLA2 phosphorylation in CECs. At the same time, only AbRAGE, but not NADPH oxidase inhibitor or ROS scavenger, inhibited the ERK1/2 pathway and cPLA2 phosphorylation in primary astrocytes. Therefore, this study demonstrates that NADPH oxidase complex assembly and ROS production are not required for Aβ42 binding to RAGE at astrocytic surface leading to sequential phosphorylation of ERK1/2 and cPLA2, and suggests the presence of two different RAGE-dependent downstream pathways in the CECs and astrocytes.

The erythrocyte's spectrin-actin membrane skeleton is directly shown to be capable of sustaining large, anisotropic strains. Photobleaching of an approximately 1-micrometer stripe in rhodamine phalloidin-labeled actin appears stable up to at least 37 degrees C, and is used to demonstrate large in-surface stretching during elastic deformation of the skeleton. Principal extension or stretch ratios of at least approximately 200% and contractions down to approximately 40%, both referenced to an essentially undistorted cell, are visually demonstrated in micropipette-imposed deformation. Such anisotropic straining is seen to be consistent at a qualitative level with now classic analyses (Evans. 1973. Biophys. J. 13:941-954) and is generally nonhomogeneous though axisymmetric down to the submicron scale. Local, direct measurements of stretching prove quantitatively consistent (within approximately 10%) with integrated estimates that are based simply on a measured relative density distribution of actin. The measurements are also in close agreement with direct computation of mean spectrin chain extension in full statistical mechanical simulations of a coarse-grained network held in a micropipette. Finally, as a cell thermally fragments near approximately 48 degrees C, the patterned photobleaching demonstrates a destructuring of the surface network in a process that is more readily attributable to transitions in spectrin than in F-actin.

Oxidative stress and inflammation are important processes in the progression of Alzheimer's disease (AD). Recent studies have implicated the role of amyloid β-peptides (Aβ) in mediating these processes. In astrocytes, oligomeric Aβ induces the assembly of NADPH oxidase complexes resulting in its activation to produce anionic superoxide. Aβ also promotes production of proinflammatory factors in astrocytes. Since low energy laser has previously been reported to attenuate oxidative stress and inflammation in biological systems, the objective of this study was to examine whether this type of laser light was able to abrogate the oxidative and inflammatory responses induced by Aβ. Primary rat astrocytes were exposed to Helium-Neon laser (λ=632.8 nm), followed by the treatment with oligomeric Aβ. Primary rat astrocytes were used to measure Aβ-induced production of superoxide anions using fluorescence microscopy of dihydroethidium (DHE), assembly of NADPH oxidase subunits by the colocalization between the cytosolic p47 phox subunit and the membrane gp91 phox subunit using fluorescent confocal microscopy, phosphorylation of cytosolic phospholipase A 2 (cPLA 2 ), and expressions of pro-inflammatory factors including interleukin-1β (IL-1β) and inducible nitric-oxide synthase (iNOS) using Western blot Analysis. Our data showed that laser light at 632.8 nm suppressed Aβ-induced superoxide production, colocalization between NADPH oxidase gp91 phox and p47 phox subunits, phosphorylation of cPLA 2 , and the expressions of IL-1β and iNOS in primary astrocytes. We demonstrated for the first time that 632.8 nm laser was capable of suppressing cellular pathways of oxidative stress and inflammatory responses critical in the pathogenesis in AD. This study should prove to provide the groundwork for further investigations for the potential use of laser therapy as a treatment for AD.

The temperature-dependent hydration of several pure, net-neutral membranes was studied by spectroscopic shifts of the amphiphilic probe 6-dodecanoyl-2-(dimethylamino)-naphthalene (LAURDAN). A calibration scale for local polarity was first established with LAURDAN in various organic solvents. For phosphatidylcholine membranes above the gel-phase temperature, the log(local polarity) was found to be inversely related to the bending-renormalized area elastic moduli. A novel self-assembled polymer membrane broadens the correlation, and the absence of any discontinuity in local polarity with temperature indicates that the polymer membrane is in a suitable fluid phase. Separate correlations between the log(hydraulic permeability × membrane thickness) and the bending modulus, but not the area elastic moduli, strongly suggest that local bending fluctuations of a membrane are coupled to membrane hydration. The results identify the importance of a collective response, membrane flexure, to a molecular-scale property, specifically, hydration.