The Three-Dimensional Distribution of αA-Crystalline in Rat Lenses and Its Possible Relation to Transparency

Lens transparency depends on the accumulation of massive quantities (600–800 mg/ml) of twelve primary crystallines and two truncated crystallines in highly elongated “fiber” cells. Despite numerous studies, major unanswered questions are how this heterogeneous group of proteins becomes organized to bestow the lens with its unique optical properties and how it changes during cataract formation. Using novel methods based on conical tomography and labeling with antibody/gold conjugates, we have profiled the 3D-distribution of the αA-crystalline in rat lenses at ∼2 nm resolutions and three-dimensions. Analysis of tomograms calculated from lenses labeled with anti-αA-crystalline and gold particles (∼3 nm and ∼7 nm diameter) revealed geometric patterns shaped as lines, isosceles triangles and polyhedrons. A Gaussian distribution centered at ∼7.5 nm fitted the distances between the ∼3 nm diameter gold conjugates. A Gaussian distribution centered at ∼14 nm fitted the Euclidian distances between the smaller and the larger gold particles and another Gaussian at 21–24 nm the distances between the larger particles. Independent of their diameters, tethers of 14–17 nm in length connected files of gold particles to thin filaments or clusters to ∼15 nm diameter “beads.” We used the information gathered from tomograms of labeled lenses to determine the distribution of the αA-crystalline in unlabeled lenses. We found that αA-crystalline monomers spaced ∼7 nm or αA-crystalline dimers spaced ∼15 nm center-to-center apart decorated thin filaments of the lens cytoskeleton. It thus seems likely that lost or gain of long-range order determines the 3D-structure of the fiber cell and possible also cataract formation

High p CO 2 promotes coral primary production

While research on ocean acidification (OA) impacts on coral reefs has focused on calcification, relatively little is known about effects on coral photosynthesis and respiration, despite these being among the most plastic metabolic processes corals may use to acclimatize to adverse conditions. Here, we present data collected between 2016 and 2018 at three natural CO2 seeps in Papua New Guinea where we measured the metabolic flexibility (i.e. in hospite photosynthesis and dark respiration) of 12 coral species. Despite some species-specific variability, metabolic rates as measured by net oxygen flux tended to be higher at high pCO(2) (ca 1200 mu atm), with increases in photosynthesis exceeding those of respiration, suggesting greater productivity of Symbiodiniaceae photosynthesis in hospite, and indicating the potential for metabolic flexibility that may enable these species to thrive in environments with high pCO(2). However, laboratory and field observations of coral mortality under high CO2 conditions associated with coral bleaching suggests that this metabolic subsidy does not result in coral higher resistance to extreme thermal stress. Therefore, the combined effects of OA and global warming may lead to a strong decrease in coral diversity despite the stimulating effect on coral productivity of OA alone

Diverse Roles of Eph/ephrin Signaling in the Mouse Lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells

Atomic-Resolution Simulations Predict a Transition State for Vesicle Fusion Defined by Contact of a Few Lipid Tails

Membrane fusion is essential to both cellular vesicle trafficking and infection by enveloped viruses. While the fusion protein assemblies that catalyze fusion are readily identifiable, the specific activities of the proteins involved and nature of the membrane changes they induce remain unknown. Here, we use many atomic-resolution simulations of vesicle fusion to examine the molecular mechanisms for fusion in detail. We employ committor analysis for these million-atom vesicle fusion simulations to identify a transition state for fusion stalk formation. In our simulations, this transition state occurs when the bulk properties of each lipid bilayer remain in a lamellar state but a few hydrophobic tails bulge into the hydrophilic interface layer and make contact to nucleate a stalk. Additional simulations of influenza fusion peptides in lipid bilayers show that the peptides promote similar local protrusion of lipid tails. Comparing these two sets of simulations, we obtain a common set of structural changes between the transition state for stalk formation and the local environment of peptides known to catalyze fusion. Our results thus suggest that the specific molecular properties of individual lipids are highly important to vesicle fusion and yield an explicit structural model that could help explain the mechanism of catalysis by fusion proteins

Localization and trafficking of aquaporin 2 in the kidney

Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood–brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2

Connexin channels and phospholipids: association and modulation

<p>Abstract</p> <p>Background</p> <p>For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.</p> <p>Results</p> <p>Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.</p> <p>Conclusion</p> <p>This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.</p

The structure of the cytoplasm of lens fibers as determined by conical tomography

Studies using conventional electron microscopy describe the cytoplasm of lens fiber cells as having essentially an amorphous structure. We hypothesized that significant structural detail might have been lost as a result of projecting the entire thickness of the section (50\u2013100 nm) onto a single plane (the \u2018\u2018projection artifact\u2019\u2019). To test this hypothesis, we studied the 3D-structure of rat lens cortical fibers before and after extracting the \u2018\u2018soluble\u2019\u2019 crystallins with low ionic strength buffers to make \u2018\u2018ghosts.\u2019\u2019 Tomographic series in conical geometry were collected at 55 tilts and by 5 rotations until completing a 360 turn by low dose methods. They were aligned using fiduciary points, reconstructed with the weighted back projection algorithm and refined by projection matching. Analysis of the 3D-maps included semiautomatic density segmentation using a computer program based on the watershed algorithm. We found that the cytoplasm of cortical fibers, though appearing amorphous in regions of the highest density, was in fact comprised of an ordered structure resembling a \u2018\u2018clustered matrix.\u2019\u2019 The matrix was comprised of thin (w6 nm diameter) filaments bent sharply at 110\u2013120 angles and studded with cubeshaped particles (the \u2018\u2018beaded\u2019\u2019 filaments). In cortical fibers, the particles measured a\ubc 14 2, b\ubc 13 2 and c\ubc 10 2.4 nm (n\ubc 30, mean SD) and were spaced at distances measuring 27.5 2.4 nm apart (n \ubc 8, mean SD), center-to-center. The matrix was formed as \u2018\u2018beaded\u2019\u2019 filaments, bound to clusters of \u2018\u2018soluble\u2019\u2019 proteins, crossed each other at nearly perpendicular angles. The matrix also made contact with the plasma membrane at a large number of distinct regions. We thus concluded that the cytoplasm of cortical lens fibers is comprised of a cytoskeletal matrix of \u2018\u2018beaded\u2019\u2019 filaments that organize the \u2018\u2018soluble\u2019\u2019 crystallins in separate regions. The association of this matrix with the plasma membrane allows the lens to maintain its structural integrity, while its association with crystallins yields its long-term transparency. Loss of either function likely would play a significant role in cataract formation

Conical Electron Tomography of a Chemical Synapse: Polyhedral Cages Dock Vesicles to the Active Zone

In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical \u201caverage\u201d synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of 3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct \u201csynaptic units\u201d comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured 60 nm in diameter, with a density of 44/ m2 and were associated with vesicles at the active zone (\u201cfirst tier\u201d). Vesicles in this first-tier position represented 7.5% of the total number of vesicles in the terminal and were contiguous, hemifused ( 4% of total), or fully fused ( 0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release