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Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.
Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
In hovering flight, hummingbirds reverse the angle of attack of their wings through pitch reversal in order to generate aerodynamic lift during both downstroke and upstroke. In addition, the wings may pitch during translation to further enhance lift production. It is not yet clear whether these pitching motions are caused by the wing inertia or actuated through the musculoskeletal system. Here we perform a computational analysis of the pitching dynamics by incorporating the realistic wing kinematics to determine the inertial effects. The aerodynamic effect is also included using the pressure data from a previous three-dimensional computational fluid dynamics simulation of a hovering hummingbird. The results show that like many insects, pitch reversal of the hummingbird is, to a large degree, caused by the wing inertia. However, actuation power input at the root is needed in the beginning of pronation to initiate a fast pitch reversal and also in mid-downstroke to enable a nose-up pitching motion for lift enhancement. The muscles on the wing may not necessarily be activated for pitching of the distal section. Finally, power analysis of the flapping motion shows that there is no requirement for substantial elastic energy storage or energy absorption at the shoulder joint.
A three-dimensional computational fluid dynamics simulation is performed for a ruby-throated hummingbird (Archilochus colubris) in hovering flight. Realistic wing kinematics are adopted in the numerical model by reconstructing the wing motion from high-speed imaging data of the bird. Lift history and the three-dimensional flow pattern around the wing in full stroke cycles are captured in the simulation. Significant asymmetry is observed for lift production within a stroke cycle. In particular, the downstroke generates about 2.5 times as much vertical force as the upstroke, a result that confirms the estimate based on the measurement of the circulation in a previous experimental study. Associated with lift production is the similar power imbalance between the two half strokes. Further analysis shows that in addition to the angle of attack, wing velocity and surface area, drag-based force and wing-wake interaction also contribute significantly to the lift asymmetry. Though the wing-wake interaction could be beneficial for lift enhancement, the isolated stroke simulation shows that this benefit is buried by other opposing effects, e.g. presence of downwash. The leading-edge vortex is stable during the downstroke but may shed during the upstroke. Finally, the full-body simulation result shows that the effects of wing-wing interaction and wing-body interaction are small.
© 2014 The Author(s) Published by the Royal Society. All rights reserved.
During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.
Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.
Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions.
Delta and Notch are required for partitioning of vein and intervein cell fates within the provein during Drosophila metamorphosis. We find that partitioning of these fates is dependent on Delta-mediated signalling from 22 to 30 hours after puparium formation at 25 degrees C. Within the provein, Delta is expressed more highly in central provein cells (presumptive vein cells) and Notch is expressed more highly in lateral provein cells (presumptive intervein cells). Accumulation of Notch in presumptive intervein cells is dependent on Delta signalling activity in presumptive vein cells and constitutive Notch receptor activity represses Delta accumulation in presumptive vein cells. When Delta protein expression is elevated ectopically in presumptive intervein cells, complementary Delta and Notch expression patterns in provein cells are reversed, and vein loss occurs because central provein cells are unable to stably adopt the vein cell fate. Our findings imply that Delta-Notch signalling exerts feedback regulation on Delta and Notch expression during metamorphic wing vein development, and that the resultant asymmetries in Delta and Notch expression underlie the proper specification of vein and intervein cell fates within the provein.