Direct evidence for interphase chromosome movement during the mid-blastula transition in Drosophila

AbstractIn Drosophila, several genetic phenomena are most easily explained by a model in which homologous chromosomes pair, at least transiently, and use regulatory information present on only one homolog to pattern expression from both homologs [1–3]. To accomplish pairing of sites on different chromosomes, there must be a mechanism by which communication between homologs is facilitated. However, except in the case of meiotic prophase, directed, rapid chromosomal movement has not yet been observed. Some studies suggest that chromosomes are relatively immobile during interphase [4,5], while others suggest that chromatin can reposition during interphase [6–8] and may be free to undergo substantial Brownian motion [9]. Using high-resolution, three-dimensional imaging techniques, we determined directly the structure and nuclear location of eleven different loci, both active and inactive, in embryos at cycle 14, the mid-blastula transition. We show that during a single interphase, portions of chromosomes moved in a cell-cycle-specific, directed fashion, independently and over long distances. All eleven regions showed movement, although the genes closer to the centromere moved faster (0.7 μm/minute) and over long distances (5–10 μm), whereas those nearer the telomere expanded in the same place and became oriented along the nuclear axis. Gene motion was independent of replication, transcription and changes in nuclear shape. Because individual genes on the same chromosome move independently, the movement is unlikely to be mediated by centromeres, Brownian motion or random drift and must be caused by an active mechanism

Differential mitochondrial roles for α-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation

Mitochondria are highly dynamic organelles with strict quality control processes that maintain cellular homeostasis. Within axons, coordinated cycles of fission-fusion mediated by dynamin related GTPase protein (DRP1) and mitofusins (MFN), together with regulated motility of healthy mitochondria anterogradely and damaged/oxidized mitochondria retrogradely, control mitochondrial shape, distribution and size. Disruption of this tight regulation has been linked to aberrant oxidative stress and mitochondrial dysfunction causing mitochondrial disease and neurodegeneration. Although pharmacological induction of Parkinson’s disease (PD) in humans/animals with toxins or in mice overexpressing α-synuclein (α-syn) exhibited mitochondrial dysfunction and oxidative stress, mice lacking α-syn showed resistance to mitochondrial toxins; yet, how α-syn influences mitochondrial dynamics and turnover is unclear. Here, we isolate the mechanistic role of α-syn in mitochondrial homeostasis in vivo in a humanized Drosophila model of Parkinson’s disease (PD). We show that excess α-syn causes fragmented mitochondria, which persists with either truncation of the C-terminus (α-syn1–120) or deletion of the NAC region (α-synΔNAC). Using in vivo oxidation reporters Mito-roGFP2-ORP1/GRX1 and MitoTimer, we found that α-syn-mediated fragments were oxidized/damaged, but α-syn1–120-induced fragments were healthy, suggesting that the C-terminus is required for oxidation. α-syn-mediated oxidized fragments showed biased retrograde motility, but α-syn1–120-mediated healthy fragments did not, demonstrating that the C-terminus likely mediates the retrograde motility of oxidized mitochondria. Depletion/inhibition or excess DRP1-rescued α-syn-mediated fragmentation, oxidation, and the biased retrograde motility, indicating that DRP1-mediated fragmentation is likely upstream of oxidation and motility changes. Further, excess PINK/Parkin, two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, rescued α-syn-mediated membrane depolarization, oxidation and cell death in a C-terminus-dependent manner, suggesting a functional interaction between α-syn and PINK/Parkin. Taken together, our findings identify distinct roles for α-syn in mitochondrial homeostasis, highlighting a previously unknown pathogenic pathway for the initiation of PD.Fil: Krzystek, Thomas J.. State University of New York; Estados UnidosFil: Banerjee, Rupkatha. State University of New York; Estados UnidosFil: Thurston, Layne. State University of New York; Estados UnidosFil: Huang, JianQiao. State University of New York; Estados UnidosFil: Swinter, Kelsey. State University of New York; Estados UnidosFil: Rahman, Saad Navid. State University of New York; Estados UnidosFil: Falzone, Tomas Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Gunawardena, Shermali. State University of New York; Estados Unido

Simulation Study of an LWFA-based Electron Injector for AWAKE Run 2

The AWAKE experiment aims to demonstrate preservation of injected electron beam quality during acceleration in proton-driven plasma waves. The short bunch duration required to correctly load the wakefield is challenging to meet with the current electron injector system, given the space available to the beamline. An LWFA readily provides short-duration electron beams with sufficient charge from a compact design, and provides a scalable option for future electron acceleration experiments at AWAKE. Simulations of a shock-front injected LWFA demonstrate a 43 TW laser system would be sufficient to produce the required charge over a range of energies beyond 100 MeV. LWFA beams typically have high peak current and large divergence on exiting their native plasmas, and optimisation of bunch parameters before injection into the proton-driven wakefields is required. Compact beam transport solutions are discussed.Comment: Paper submitted to NIMA proceedings for the 3rd European Advanced Accelerator Concepts Workshop. 4 pages, 3 figures, 1 table Changes after revision: Figure 2: figures 2 and 3 of the previous version collated with plots of longitudinal electric field Line 45: E_0 = 96 GV/m Lines 147- 159: evaluation of beam loading made more accurate Lines 107 - 124: discussion of simulation geometry move

Organically Modified Silica Nanoparticles Are Biocompatible and Can Be Targeted to Neurons In Vivo

The application of nanotechnology in biological research is beginning to have a major impact leading to the development of new types of tools for human health. One focus of nanobiotechnology is the development of nanoparticle-based formulations for use in drug or gene delivery systems. However most of the nano probes currently in use have varying levels of toxicity in cells or whole organisms and therefore are not suitable for in vivo application or long-term use. Here we test the potential of a novel silica based nanoparticle (organically modified silica, ORMOSIL) in living neurons within a whole organism. We show that feeding ORMOSIL nanoparticles to Drosophila has no effect on viability. ORMOSIL nanoparticles penetrate into living brains, neuronal cell bodies and axonal projections. In the neuronal cell body, nanoparticles are present in the cytoplasm, but not in the nucleus. Strikingly, incorporation of ORMOSIL nanoparticles into the brain did not induce aberrant neuronal death or interfered with normal neuronal processes. Our results in Drosophila indicate that these novel silica based nanoparticles are biocompatible and not toxic to whole organisms, and has potential for the development of long-term applications

Interphase chromosome movement during the midblastula transition in Drosophila melanogaster

Eukaryotic chromatin is functionally active only in the interphase nucleus. Indirectly we know that global chromatin changes occur, such that gene expression and replication proceed. I undertook to directly observe the structural changes of interphase chromatin, at a time in Drosophila embryogenesis when many nuclear processes were just beginning to be established. I reasoned that cycle 14 was the ideal time in which to observe chromatin changes as a result of functional processes. During this embryonic stage cellular processes shifts from maternal to zygotic control. Chromosomes also undergo significant changes. To infer the native structure of chromatin, I developed an ultra-sensitive two colour in situ hybridization (FISH) technique and established its limits of resolution. Combining ultrasensitive FISH, with high resolution three-dimensional imaging techniques, I can visualize directly the compaction, position and orientation of genes within the interphase nucleus. I first characterized to a greater extent the chromatin changes in the Notch gene during the mid-blastula transition. I observe that the Notch gene decondense as the embryo ages in cycle 14. I further localized both individually and simultaneously, a variety of genes on the three large chromosomes of Drosophila. I observe that during a single interphase, portions of chromosomes move in a cell cycle specific and directed fashion; both independently and over long distances. From these results I conclude that global chromatin changes occur during interphase. I suggest that chromatin is organized beyond the Rab1 orientation such that the position of the gene on the chromosome allows loci to move independently within the active interphase nucleus. I propose a model for chromatin organization within the Drosophila interphase nucleus. Within the Rab1 order, higher-order chromatin is organized into loop domains, ranging in size from 5--100s of kb. I postulate that loop domains that are centromere proximal are small in size, 5-50 kb, while those centromere distal are larger, often greater than 100 kbs, consistent with the observation that the centromere proximal histone gene cluster is arranged in a 5 kb loop (Mirkovitch et al., 1984), while the Notch gene which is near the telomere, is part of a larger loop (Gunawardena et al., 1999a). The loops are attached to each other by a chromosomal backbone structure. My observations demonstrate that interphase nuclear function is superimposed and permitted on this loop-backbone chromatin organization, such that gene movement occurs (Gunawardena et al., 1999b)

A 3-dimensional structural analysis of diploid chromosomes

In this study, we are looking at the 3-dimensional chromosome structure of interphase diploid nuclei of Drosophila melanogaster. The goal is to determine the higher order structure of interphase chromosomes in these nuclei. Higher order structures include those structures larger than the 30nm fiber. Over the years, several general models for higher order chromosome structures have been presented. We look at three popular models for the organization of chromatin during embryogenesis, as each of these models make predictions that can be tested using high resolution in situ hybridization and image processing techniques. For this study we are using the Notch gene for in situ hybridization to embryos in cycles 10-14.Our preliminary results are inconsistent with the radial loop model. It appears that the chromatin might be arranged in folds of 30 and 10nm fibers. We also observe a difference in chromatin structure as the embryo gets older. As the Notch gene is being transcribed during cycle 14 we observe a puffing event. In this study we hope to expand on these observations and present further areas that need to be explored in order to conclusively distinguish these phenomena during early embryogenesis.Digitized from a paper copy provided by the Genetics Graduate Interdisciplinary Program (GIDP)

Axonal Transport and Neurodegeneration: How Marine Drugs Can Be Used for the Development of Therapeutics

Unlike virtually any other cells in the human body, neurons are tasked with the unique problem of transporting important factors from sites of synthesis at the cell bodies, across enormous distances, along narrow-caliber projections, to distally located nerve terminals in order to maintain cell viability. As a result, axonal transport is a highly regulated process whereby necessary cargoes of all types are packaged and shipped from one end of the neuron to the other. Interruptions in this finely tuned transport have been linked to many neurodegenerative disorders including Alzheimer’s (AD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) suggesting that this pathway is likely perturbed early in disease progression. Therefore, developing therapeutics targeted at modifying transport defects could potentially avert disease progression. In this review, we examine a variety of potential compounds identified from marine aquatic species that affect the axonal transport pathway. These compounds have been shown to function in microtubule (MT) assembly and maintenance, motor protein control, and in the regulation of protein degradation pathways, such as the autophagy-lysosome processes, which are defective in many degenerative diseases. Therefore, marine compounds have great potential in developing effective treatment strategies aimed at early defects which, over time, will restore transport and prevent cell death