New mechanism of membrane fusion

We have carried out Monte Carlo simulation of the fusion of bilayers of single chain amphiphiles which show phase behavior similar to that of biological lipids. The fusion mechanism we observe is very different from the ``stalk'' hypothesis. Stalks do form on the first stage of fusion, but they do not grow radially to form a hemifused state. Instead, stalk formation destabilizes the membranes and results in hole formation in the vicinity of the stalks. When holes in each bilayer nucleate spontaneously next to the same stalk, an incomplete fusion pore is formed. The fusion process is completed by propagation of the initial connection, the stalk, along the edges of the aligned holes.Comment: 4 pages, 3 figure

Dynein structure and power stroke

Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke

Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

Determining the mechanism by which transfer RNAs (tRNAs) rapidly and precisely transit through the ribosomal A, P and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pre-translocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium towards the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within post-translocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA

Dorsoventral patterning of the Xenopus eye involves differential temporal changes in the response of optic stalk and retinal progenitors to Hh signalling

Background: Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, promoting optic stalk and ventral retinal fates and repressing dorsal retinal identity. There has been limited analysis, however, of the critical window during which Hh molecules control eye polarity and of the temporal changes in the responsiveness of eye cells to these signals. Results: In this study, we used pharmacological and molecular tools to perform stage-specific manipulations of Hh signalling in the developing Xenopus eye. In gain-of-function experiments, most of the eye was sensitive to ventralization when the Hh pathway was activated starting from gastrula/neurula stages. During optic vesicle stages, the dorsal eye became resistant to Hh-dependent ventralization, but this pathway could partially upregulate optic stalk markers within the retina. In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the ventral retina and the optic stalk, while the effects of Hh inhibition during optic vesicle stages were limited to the reduction of optic stalk size. Conclusions: Our results suggest the existence of two competence windows during which the Hh pathway differentially controls patterning of the eye region. In the first window, between the neural plate and the optic vesicle stages, Hh signalling exerts a global influence on eye dorsoventral polarity, contributing to the specification of optic stalk, ventral retina and dorsal retinal domains. In the second window, between optic vesicle and optic cup stages, this pathway plays a more limited role in the maintenance of the optic stalk domain. We speculate that this temporal regulation is important to coordinate dorsoventral patterning with morphogenesis and differentiation processes during eye development

Radar response from vegetation with nodal structure

Radar images from the SEASAT synthetic aperture radar (SAR) produced unusually high returns from corn and sorghum fields, which seem to indicate a correlation between nodal separation in the stalk and the wavelength of the radar. These images also show no difference in return from standing or harvested corn. Further investigation using images from the Shuttle Imaging Radar (SIR-A) substantiated these observations and showed a degradation of the high return with time after harvest. From portions of corn and sweet sorghum stalks that were sampled to measure stalk water content, it was determined that near and after maturity the water becomes more concentrated in the stalk nodes. The stalk then becomes a linear sequence of alternating dielectrics as opposed to a long slender cylinder with uniform dielectric properties

A New Mechanism of Model Membrane Fusion Determined from Monte Carlo Simulation

We have carried out extensive Monte Carlo simulations of the fusion of tense apposed bilayers formed by amphiphilic molecules within the framework of a coarse grained lattice model. The fusion pathway differs from the usual stalk mechanism. Stalks do form between the apposed bilayers, but rather than expand radially to form an axial-symmetric hemifusion diaphragm of the trans leaves of both bilayers, they promote in their vicinity the nucleation of small holes in the bilayers. Two subsequent paths are observed: (i) The stalk encircles a hole in one bilayer creating a diaphragm comprised of both leaves of the other intact bilayer, and which ruptures to complete the fusion pore. (ii) Before the stalk can encircle a hole in one bilayer, a second hole forms in the other bilayer, and the stalk aligns and encircles them both to complete the fusion pore. Both pathways give rise to mixing between the cis and trans leaves of the bilayer and allow for transient leakage.Comment: revised version, accepted to Biophys. J. (11 figures

Myosin IIA-mediated forces regulate multicellular integrity during vascular sprouting

Angiogenic sprouting is a critical process involved in vascular network formation within tissues. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell-cell junctions, forming patent structures that support blood flow. Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell-cell dissociation primarily between tip and stalk cells. Closer examination of cell-cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell-cell adhesions during collective motility.R01 EB000262 - NIBIB NIH HHS; R01 HL115553 - NHLBI NIH HHSPublished versio

Computational Screening of Tip and Stalk Cell Behavior Proposes a Role for Apelin Signaling in Sprout Progression

Angiogenesis involves the formation of new blood vessels by sprouting or splitting of existing blood vessels. During sprouting, a highly motile type of endothelial cell, called the tip cell, migrates from the blood vessels followed by stalk cells, an endothelial cell type that forms the body of the sprout. To get more insight into how tip cells contribute to angiogenesis, we extended an existing computational model of vascular network formation based on the cellular Potts model with tip and stalk differentiation, without making a priori assumptions about the differences between tip cells and stalk cells. To predict potential differences, we looked for parameter values that make tip cells (a) move to the sprout tip, and (b) change the morphology of the angiogenic networks. The screening predicted that if tip cells respond less effectively to an endothelial chemoattractant than stalk cells, they move to the tips of the sprouts, which impacts the morphology of the networks. A comparison of this model prediction with genes expressed differentially in tip and stalk cells revealed that the endothelial chemoattractant Apelin and its receptor APJ may match the model prediction. To test the model prediction we inhibited Apelin signaling in our model and in an \emph{in vitro} model of angiogenic sprouting, and found that in both cases inhibition of Apelin or of its receptor APJ reduces sprouting. Based on the prediction of the computational model, we propose that the differential expression of Apelin and APJ yields a "self-generated" gradient mechanisms that accelerates the extension of the sprout.Comment: 48 pages, 10 figures, 8 supplementary figures. Accepted for publication in PLoS ON

Fermentation of stalk juices from different Nigerian sorghum cultivars to ethanol

For improved production of ethanol from sorghum stalk juice fermentation, cultivation location and cultivar type are important factors to consider. In the present study, SSV2 and KSV8 sorghum cultivars were cultivated in Kano and Kaduna states in Nigeria that exhibit notably different rain precipitation and diurnal temperatures. The crude stalk juices (without pre-treatment or nutrient supplementation) were extracted from these sorghum samples and fermented with a distiller’s strain of the yeast, Saccharomyces cerevisiae. Sugar consumption and alcohol production were determined by HPLC and GC-MS, respectively. When it was grown in the Kaduna site, SSV2 was identified as the highest yielding sorghum cultivar from which we extracted the maximum levels of extractable sugars (161.50 g l-1 ) that yielded favourable ethanol levels of 80.56 g l-1 following fermentation. Our findings show that relatively colder and wetter cultivation sites are preferred for sorghum stalk juice destined for bioethanol production.<br/