Spektrales konfokales Laserscanning-Mikroskop
Final Report Abstract
1. Institut für Anatomie und Zellbiologie (Prof. K. Endlich) Im Berichtszeitraum wurden an dem cLSM kultivierte Podozyten und der Pronephros von Zebrafischlarven visualisiert. An kultivierten Podozyten untersuchten wir die HB-EGF induzierte Bildung von spezifischen dorsalen Aktin-Strukturen (RiLiS-Ring-Like Actin Structures), die wahrscheinlich für die Migration von Podozyten in der rapid progressiven Glomerulonephritis verantwortlich sind. Hier war die confocale Laser-scan Mikroskopie jedoch nicht die tragende Hauptarbeitsmethode. Des Weiteren untersuchten wir an kultivierten Podozyten Zellausläufer (TAPs-Thin Arborized Projections), die sich unter Überexpression des essenziellen Podozyten-Proteins CD151 bilden. An Zebrafischlarven visualisierten wir den Glomerulus des Pronephros unter Kontrollbedingungen und nach Knockdown des essenziellen Podozyten-Proteins Myh9. In diesen beiden letzten Arbeiten spielte die Konfokalmikroskopie eine wesentliche Rolle. 2. AG Physiologie und Biochemie der Tiere (Prof. J. Hildebrandt): Cell biological effects of poreforming bacterial toxins on human airway epithelial cells In intact airways, inhaled bacteria do not get in direct contact with the apical surfaces of epithelial cells due to the presence of a thick mucus layer covering the ciliated epithelium. The ciliary activity results in a steady movement of mucus toward the throat resulting in mechanical clearance of the airways. Dysfunction of mucociliary clearance may occur in airways whose functions are disturbed by acute (e.g. viral infection) or chronic diseases (e.g. cystic fibrosis). In such cases, bacteria like Staphylococcus aureus (S. aureus), which occurs in the nasal cavities of humans, may stay on the mucus surface long enough to form colonies and secrete soluble virulence-associated factors as hemolysin A (Hla). Such factors, in contrast to the bacteria themselves, may readily pass through the mucus layer by diffusion. Even in sublethal concentrations, such factors may affect the barrier function of the epithelium as indicated by changes in cell layer integrity observed previously in Hla-treated endothelial cells. In the airways, such irritations may result in disruption of the intact mucus layer allowing physical contact of bacteria with the apical cell surface, bacterial adherence, internalization of bacteria into the host cell and airway infection. That Hla is actually produced by S. aureus even in minor infections is illustrated by the facts that patients generate antibodies against Hla and that vaccination against Hla protects animals from developing S. aureus-mediated pneumonia. Hla forms heptameric pores in the plasma membranes of eukaryotic cells. Addition of recombinant Hla (rHla) to the extracellular medium of cultured airway epithelial cells induces elevations in the concentration of free calcium ions in the cytosol of epithelial cells and may increase membrane permeability for monovalent cations. Such alterations may affect ciliary beating frequency, fluid and mucus secretion as well as cell survival or may result in cell shape changes. On the other hand, such changes may activate defensive actions of the epithelium or secretion of pro-inflammatory cytokines and chemokines. Treatment of primary or immortalized human airway epithelial cells (16HBE14o-, S9) or alveolar cancer cells (A549) with rHla induces alterations in cell shape and formation of paracellular gaps in the cell layer. Semi-quantitative Western blotting using extracts of freshly isolated airway tissue (nasal epithelium) or 16HBE14o-model cells revealed that phosphorylation levels of focal adhesion kinase (Fak) and paxillin were altered upon treatment of tissue or cells with rHla. Immune fluorescence analyses using a confocal microscope showed that rHlatreatment of 16HBE14o- cells results in losses of vinculin and paxillin from focal contacts and a net reduction in the number of focal contacts. The actin cytoskeleton was strongly remodelled. We conclude that treatment of cells with rHla activates Src- and Fak-mediated intracellular signalling which accelerates focal contact turnover and prevents newly formed focal contacts (focal complexes) from maturation to focal adhesions. The inability of rHla-treated cells to form stable focal adhesions may be one factor that contributes to gap formation in the cell layer. In vivo, such changes may disturb the defensive barrier function of the airway epithelium and may facilitate lung infections by S. aureus. 3. AG Cytologie und Evolutionsbiologie (Prof. S. Harzsch): Evolution of the olfactory system in Myriapoda Originating from a marine ancestor, the myriapods most likely invaded land independently of the hexapods. As these two evolutionary lineages conquered land in parallel but separately, we are interested in comparing the myriapod chemosensory system to that of hexapods to gain insights into possible adaptations for olfaction in air. Our projects connects to a previous analysis of the brain and behavior of the chilopod (centipede) Scutigera coleoptrata in which we demonstrated that these animals do respond to volatile substances and analyzed the structure of their central olfactory pathway. In the current project we examined the architecture of the deutocerebral brain areas (which process input from the antennae) in seven additional representatives of the Chilopoda, covering all major subtaxa, by histology, confocal laser-scan microscopy, and 3D reconstruction. We found that in all species that we studied the majority of antennal afferents target two separate neuropils, the olfactory lobe (chemosensory, composed of glomerular neuropil compartments) and the corpus lamellosum (mechanosensory). The numbers of olfactory glomeruli in the different chilopod taxa ranged from ca. 35 up to ca. 90 and the shape of the glomeruli ranged from spheroid across ovoid or drop-shape to elongate. A split of the afferents from the (first) pair of antennae into separate chemosensory and mechanosensory components is also typical for Crustacea and Hexapoda, but this set of characters is absent in Chelicerata. We suggest that this character set strongly supports the Mandibulata hypothesis (Myriapoda + (Crustacea + Hexapoda)) as opposed to the Myriochelata concept (Myriapoda + Chelicerata). 4. AG Cytologie und Evolutionsbiologie (Prof. S. Harzsch): The chemical senses of marine isopod crustaceans Representatives of at least six crustacean taxa managed to establish a terrestrial life style during their evolutionary history and the Oniscidea (Isopoda) are currently held as the most successfully terrestrialized malacostracan crustaceans. The brain architecture of terrestrial isopods is fairly well understood and studies on this field suggest that the evolutionary transition from sea to land in isopods coincided with a considerable size reduction and functional loss of their first pair of antennae and associated brain areas. This finding suggests that terrestrial isopods may have no or poor abilities to detect volatile substances but that their chemosensory ecology is most likely restricted to contact chemoreception. In this project, we explored how the brain of a marine isopod and particularly its olfactory system compares to that of terrestrial relatives. Using histochemical and immunohistochemical labeling, brightfield and confocal laser-scan microscopy, we showed that in the marine isopod Saduria entomon aesthetascs on the first pair of antennae provide input to a well defined deutocerebrum (DC). The deutocerebral chemosensory lobes (DCL) are divided into spherical neuropil compartments, the olfactory glomeruli (og). Secondary processing areas in the lateral protocerebrum (lPC) are supplied by a thin but distinct projection neuron tract (PNT) with a contralateral connection. Hence, contrary to terrestrial Isopoda, S. entomon has at least the neuronal substrate to perceive and process olfactory stimuli suggesting the originally marine isopod lineage had olfactory abilities comparable to that of other malacostracan crustaceans.
Publications
- (2011) Non-muscle myosin IIA is required for the development of the zebrafish glomerulus. Kidney Int. 80: 1055-63
Tobias Müller, Elisabeth Rumpel, Susanne Hradetzky, Frank Bollig, Henny Wegner, Antje Blumenthal, Andreas Greinacher, Karlhans Endlich, Nicole Endlich
- (2012) Comparative analysis of deutocerebral neuropils in Chilopoda (Myriapoda): implications for the evolution of the arthropod olfactory system and support for the Mandibulata concept. BMC Neuroscience 13: 1
Sombke A, Lipke E, Kenning M, Müller C, Hansson BS, Harzsch S
- (2013) Brain anatomy of the marine isopod Saduria entomon Linnaeus, 1758 (Valvifera, Isopoda) with special emphasis on the olfactory pathway. Frontiers in Neuroanatomy
Kenning M, Harzsch S
(See online at https://doi.org/10.3389/fnana.2013.00032) - (2013) Neurogenesis in an early protostome relative: progenitor cells in the ventral nerve centre of chaetognath hatchlings are arranged in a highly organized geometrical pattern. Journal of Experimental Zoology. 320(3):179-193
Perez Y, Rieger V, Martin E, Müller C, Harzsch S
- (2013) The Malacostraca (Crustacea) from a neurophylogenetic perspective: new insights from brain architecture in Nebalia herbstii Leach, 1814 (Leptostraca, Phyllocarida). Zoologischer Anzeiger 252: 319-336
Kenning M , Müller C, Wirkner CS, Harzsch S
- (2013): A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the Marbled Crayfish and the Migratory Locust: evidence for a homologous protocerebral group of neurons. Arthropod Structure & Development 42(6): 507-520
Zieger E, Bräunig P, Harzsch S
- (2014) Morphology and migration of podocytes are affected by CD151 levels. American Journal of Physiology - Renal Physiology Vol. 302, F1265-F1277
Antje Blumenthal, Jürgen Giebel, Ramesh Ummanni, Rabea Schlüter, Karlhans Endlich, Nicole Endlich
(See online at https://doi.org/10.1152/ajprenal.00468.2011) - (2014) Staphylococcus aureushemolysin A disrupts cell-matrix adhesions in human airway epithelial cells. Am J Respir Cell Mol Biol
Hermann I, Räth S, Ziesemer S, Volksdorf T, Dress RJ, Gutjahr M, Müller C, Beule AG, Hildebrandt J-P
(See online at https://doi.org/10.1165/rcmb.2014-0082OC) - (2014). Immunohistochemical and ultrastructural studies on ciliary sense organs of arrow worms (Chaetognatha). Zoomorphology 133:167–189
Müller CHG; Rieger V, Perez Y & Harzsch S
(See online at https://doi.org/10.1007/s00435-013-0211-6)
