COPI vesicle transport is a common requirement for tube expansion in Drosophila

Background

Tube expansion defects like stenoses and atresias cause devastating human diseases. Luminal expansion during organogenesis begins to be elucidated in several systems but we still lack a mechanistic view of the process in many organs. The Drosophila tracheal respiratory system provides an amenable model to study tube size regulation. In the trachea, COPII anterograde transport of luminal proteins is required for extracellular matrix assembly and the concurrent tube expansion.

Principal Findings

We identified and analyzed Drosophila COPI retrograde transport mutants with narrow tracheal tubes. γCOP mutants fail to efficiently secrete luminal components and assemble the luminal chitinous matrix during tracheal tube expansion. Likewise, tube extension is defective in salivary glands, where it also coincides with a failure in the luminal deposition and assembly of a distinct, transient intraluminal matrix. Drosophila γCOP colocalizes with cis-Golgi markers and in γCOP mutant embryos the ER and Golgi structures are severely disrupted. Analysis of γCOP and Sar1 double mutants suggests that bidirectional ER-Golgi traffic maintains the ER and Golgi compartments and is required for secretion and assembly of luminal matrixes during tube expansion.

Conclusions/Significance

Our results demonstrate the function of COPI components in organ morphogenesis and highlight the common role of apical secretion and assembly of transient organotypic matrices in tube expansion. Intraluminal matrices have been detected in the notochord of ascidians and zebrafish COPI mutants show defects in notochord expansion. Thus, the programmed deposition and growth of distinct luminal molds may provide distending forces during tube expansion in diverse organs.     

Silkworm coatomers and their role in tube expansion of posterior silkgland

Background

Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.

Methodology/Principal Findings

Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.

Conclusions/Significance

The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.     

Chemically mediated mechanical expansion of the pollen tube cell wall

Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.     

First clinical experience with the R Stent: a new highly flexible stainless steel tube intracoronary stent

BACKGROUND: Coronary stents have been used with increasing frequency and in increasingly complex coronary disease. A new 316 LVM stainless steel coronary stent, the R Stent, has been designed to provide maximum flexibility for tracking and high radial strength post-deployment. PURPOSE: To assess the clinical feasibility of the R Stent in a tertiary referral population of patients with coronary heart disease. Specific objectives are to assess the R Stent's deployment success, angiographic and procedural success (<20% residual stenosis and >TIMI 2 flow), safety (absence of complications), and 30-day clinical success (angiographic/procedural success plus no major adverse coronary events). METHODS: Between April and November 1998, stent deployment was attempted in 27 patients with stable (46%) or unstable (54%) angina pectoris who qualified for percutaneous transluminal coronary angioplasty. Eighty per cent of patients had a pre-existing history of myocardial infarction, coronary bypass surgery or percutaneous transluminal coronary angioplasty, and several of the lesions were anatomically complex (totally occluded, n 32; thrombus present, n 32; heavily calcified, n 33; ostial, n 31; >20 mm long, n 39; angulation >45 degrees, n 37). Lesions in aortocoronary saphenous vein grafts were excluded. Adjunctive medical management included intraprocedural aspirin and heparin and post-procedural aspirin and ticlopidine. After deployment, patients were followed up in the hospital and at 30 days post procedure. RESULTS: Stent deployment was achieved in 32 of 33 attempts (26 of 27 patients). There was one deployment failure in a long, calcified ostial and proximal left coronary lesion. In the 26 successful deployments, TIMI 3 flow was achieved. One other patient experienced a painless increase in creatine kinase to 375 (CK-MB of 59) at 12 h without ECG changes. At 30 days, there were no deaths, no myocardial infarctions, no subacute thromboses, no repeat interventions, no bypass surgeries and no bleeding complications. Only the patient with post-procedural CK-MB elevation experience recurrence of CCS class 2 angina within the 30 days. CONCLUSION: The R Stent is a promising new device for the treatment of complex coronary heart disease. A larger, more broadly-based study is warranted.     

Microbubble Fabrication of Concave-porosity PDMS Beads

Microbubble fabrication (by use of a fine emulsion) provides a means of increasing the surface-area-to-volume (SAV) ratio of polymer materials, which is particularly useful for separations applications. Porous polydimethylsiloxane (PDMS) beads can be produced by heat-curing such an emulsion, allowing the interface between the aqueous and aliphatic phases to mold the morphology of the polymer. In the procedures described here, both polymer and crosslinker (triethoxysilane) are sonicated together in a cold-bath sonicator. Following a period of cross-linking, emulsions are added dropwise to a hot surfactant solution, allowing the aqueous phase of the emulsion to separate, and forming porous polymer beads. We demonstrate that this method can be tuned, and the SAV ratio optimized, by adjusting the electrolyte content of the aqueous phase in the emulsion. Beads produced in this way are imaged with scanning electron microscopy, and representative SAV ratios are determined using Brunauer–Emmett–Teller (BET) analysis. Considerable variability with the electrolyte identity is observed, but the general trend is consistent: there is a maximum in SAV obtained at a specific concentration, after which porosity decreases markedly.     

Phylogeny of Itaquasconinae in the light of the evolution of the flexible pharyngeal tube in Tardigrada

The family Hypsibiidae comprises four subfamilies, among which Itaquasconinae are the most abundant in genera. Although being morphologically diverse and cosmopolitan, itaquasconins are scarcely reported and their taxonomy is entangled. Here, we present a phylogeny based on three DNA markers and morphological clues obtained from a detailed scanning electron microscopy analysis of bucco-pharyngeal apparatuses of 25 species representing seven genera. Our study revealed that (a) the polyphyletic Adropion consists of at least three evolutionary lineages, with Guidettion gen. nov. being the sister group of all remaining itaquasconins; (b) stylet supports were independently lost in Astatumen and Insulobius gen. nov.; (c) pharyngeal tube annulation can be divided into two general types, simple and complex, and is stable at the genus level, but being homoplasious (similarly to the apophyses for the insertion of stylet muscles and the shape of the claw bases), it is not useful in inferring phyletic relationships within the subfamily; and (d) Astatumen and Platicrista contain numerous similar species, challenging the current taxonomic classification; moreover, (e) three new taxa are proposed to accommodate the newly recognised lineages: Guidettion gen. nov. (the former Adropion prorsirostre group), Insulobius gen. nov. (for a new Malayan species with a mixture of Astatumen and Itaquascon traits), and Raribius gen. nov. (former Itaquascon spp. with an elongated and narrowed pharyngeal tube terminating with a spherical pharynx). The simple type of annulation is synapomorphic to Hypsibiidae, with single reversals to the uniform buccal tube in Parascon (Itaquasconinae) and in Hypsibiinae. The pharyngeal tube evolved convergently several times, and is of different character in classes Heterotardigrada and Eutardigrada.     

Transient behavior in a flexible assembly system

We consider an assembly line with m stations in series having finite-capacity buffers. Blocking occurs when buffers are full. There are M different types of products to be assembled, each with its own processing requirements. There is a production target set for each type. We give tight bounds on how long it takes such a system to reach steady state for a given cyclic schedule.     

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents^7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region¹⁰. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.Download video file.^{(51M, mov)}     

Sheared bioconvection in a horizontal tube

The recent interest in using microorganisms for biofuels is motivation enough to study bioconvection and cell dispersion in tubes subject to imposed flow. To optimize light and nutrient uptake, many microorganisms swim in directions biased by environmental cues (e.g. phototaxis in algae and chemotaxis in bacteria). Such taxes inevitably lead to accumulations of cells, which, as many microorganisms have a density different to the fluid, can induce hydrodynamic instabilites. The large-scale fluid flow and spectacular patterns that arise are termed bioconvection. However, the extent to which bioconvection is affected or suppressed by an imposed fluid flow and how bioconvection influences the mean flow profile and cell transport are open questions. This experimental study is the first to address these issues by quantifying the patterns due to suspensions of the gravitactic and gyrotactic green biflagellate alga Chlamydomonas in horizontal tubes subject to an imposed flow. With no flow, the dependence of the dominant pattern wavelength at pattern onset on cell concentration is established for three different tube diameters. For small imposed flows, the vertical plumes of cells are observed merely to bow in the direction of flow. For sufficiently high flow rates, the plumes progressively fragment into piecewise linear diagonal plumes, unexpectedly inclined at constant angles and translating at fixed speeds. The pattern wavelength generally grows with flow rate, with transitions at critical rates that depend on concentration. Even at high imposed flow rates, bioconvection is not wholly suppressed and perturbs the flow field.     

Lunatic fringe causes expansion and increased neurogenesis of trunk neural tube and neural crest populations

Both neurons and glia of the PNS are derived from the neural crest. In this study, we have examined the potential function of lunatic fringe in neural tube and trunk neural crest development by gain-of-function analysis during early stages of nervous system formation. Normally lunatic fringe is expressed in three broad bands within the neural tube, and is most prominent in the dorsal neural tube containing neural crest precursors. Using retrovirally-mediated gene transfer, we find that excess lunatic fringe in the neural tube increases the numbers of neural crest cells in the migratory stream via an apparent increase in cell proliferation. In addition, lunatic fringe augments the numbers of neurons and upregulates Delta-1 expression. The results indicate that, by modulating Notch/Delta signaling, lunatic fringe not only increases cell division of neural crest precursors, but also increases the numbers of neurons in the trunk neural crest.     

Binding mode prediction for a flexible ligand in a flexible pocket using multi-conformation simulated annealing pseudo crystallographic refinement

We describe multi-conformation simulated annealing-pseudo-crystallographic refinement (MCSA-PCR), a technique developed for predicting the binding mode of a flexible ligand in a flexible binding pocket. To circumvent the local-minimum problem efficiently, this method performs multiple independent cycles of simulated annealing with explicit solvent, "growing" the ligand in the binding pocket each time. From the ensemble of structures, a pseudo-crystallographic electron density map is calculated, and then conventional crystallographic refinement methods are used to best fit a single, optimal structure into the density map. The advantage of the MCSA-PCR method is that it provides a direct means to evaluate the accuracy and uniqueness of the calculated solution, provides a measure of ligand and protein dynamics from the refined B-factors, and facilitates comparison with X-ray crystallographic data. Here, we show that our MCSA-PCR method succeeds in predicting the correct binding mode of the VSV8 peptide to the major histocompatibility complex (MHC) receptor. Importantly, there is a significant correlation between the experimentally determined crystallographic water molecules and water density observed in the pseudo map by MCSA-PCR. Furthermore, comparison of different approaches for extracting a single, most probable structure from the calculated ensemble reveals the power of the PCR method and provides insights into the nature of the energetic landscape.     

Allocating buffer storages in a flexible manufacturing system

This article reports an approximation scheme to determine buffer capacities required to achieve the target performance level in a general flexible manufacturing system with multiple products. This work extends our previous study of sequential production lines with a single product. The manufacturing system is operated using a pull mechanism, and the performance level is measured by the average proportion of demands backlogged. Simulation experiments were performed to study the validity of the approximation scheme under various situations.     

Learning flexible sensori-motor mappings in a complex network

Given the complex structure of the brain, how can synaptic plasticity explain the learning and forgetting of associations when these are continuously changing? We address this question by studying different reinforcement learning rules in a multilayer network in order to reproduce monkey behavior in a visuomotor association task. Our model can only reproduce the learning performance of the monkey if the synaptic modifications depend on the pre- and postsynaptic activity, and if the intrinsic level of stochasticity is low. This favored learning rule is based on reward modulated Hebbian synaptic plasticity and shows the interesting feature that the learning performance does not substantially degrade when adding layers to the network, even for a complex problem.     

Phenotypically flexible sex allocation in a simultaneous hermaphrodite

Previous studies on sex allocation in simultaneous hermaphrodites have typically focused either on evolutionary or one-time, ontogenetic optimization of sex allocation, ignoring variation within an individual's lifetime. Here, we study whether hermaphrodites also possess facultative sex allocation, that is, a phenotypic flexibility, allowing them to distribute resources to either sex in an opportunistic way during their adult lifetime. We used the simultaneously hermaphroditic free-living flatworm Macrostomum lignano and raised individuals in pairs and groups of eight worms (further called octets) until sexual maturity was reached and sex allocation for the current conditions was expected to be set. Treatment groups were subsequently transferred to the alternative group size, that is, from pairs to octets or from octets to pairs, and compared to two control groups, which were transferred without changing group size. The results show that worms in treatment groups responded as expected by the local mate competition theory for simultaneous hermaphrodites: increasing group size resulted in a shift toward a more male-biased sex allocation and vice versa. These findings reveal that sex allocation in these animals is not fixed during ontogeny, but remains flexible after maturation. We argue that phenotypically flexible sex allocation in hermaphroditic animals may help us to understand the evolution and ecology of hermaphroditism.     

Thermal expansion of a protein

The thermal expansion of a protein, metmyoglobin, was investigated by analysis of the refined X-ray crystal structures at 80 and 255-300 K. On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%. The linear thermal expansion coefficient is estimated to be 115 X 10(-6) K-1. This value is more than twice as large as that of liquid water but less than that of benzene. As the temperature is raised, the internal volume change does not come from the large, atom-sized internal cavities in the structure but from an increase in the small, subatomic free volumes between atoms. The largest expansion occurs in the region of the CD and GH corners; both these regions move away from the center of the protein. The remainder of the expansion results from the lengthening of contacts between segments of secondary structure.