Biochemical and functional analysis of innexin2-containing gap junction channels during organogenesis in Drosophila [Elektronische Ressource] / vorgelegt von Hildegard Lechner

Hildegard Lechner

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Biochemical and functional analysis of
innexin2-containing gap junction channels
during organogenesis in Drosophila
Dissertation
zur
Erlangung de sDoktorgrade (s Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn
vorgelegt von
Hildegard Lechner
aus
Bonn
Bonn 2008Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheini schen
Friedrich-Wilhelms-Universität Bonn
Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn htutpn:t/e/rh ss.ulb.uni-
bonn.de/diss_online elektronisch publiziert.
Erscheinungsjahr: 2009
1. Gutachter: Prof. Dr. rer. nat. M. Hoch
2. Gutachter: Prof. Dr. rer. nat. T. Lang

T ag de r Promotion: 17.03.2009Parts of this work are published in a scientific journal:
Lechner, H,. Josten, F., Fuss, B., Bauer, R., Hoch, M. (2007). Cross regulation of inter cellular gap
junction communication and paracrine signaling pathways during organogeneDsirso soinph ila.
Dev Biol. 310: 23-34.
Other publications:
Bauer, R., Weimbs, AL.e,ch ner H,. Hoch, M. (200D6E)-.ca dherin, a core component of the
adherens junction complex modifies subcellular localization oDf rotsohep hila gap junction prote in
innexin2. Cell Commun. Adhes. 13: 103-114.
Lehmann, C., Lechner, H,. Löer, B., Knieps, M., Herrmann, S., Famulok, M., Bauer, R. ,Hoch, M.
(2006). Heteromerization of innexin gap junction proteins regulates epithelial tissue org anization in
Drosophila. Mol. Biol. Cell. 17: 1676-1685.
Bauer, R., Löer, B., Ostrowski, K., Martini, J., WeLimechbs,n eAr,. , H,. Hoch, M. (20 05).
Intercellular communication: theDr osophila innexin multiprotein family of gap junction pro teins.
Chem. Biol. 12: 515-526. Abbreviations
A.bidest Aqua bidestillata
Å Angstrom
aa Amino acid
Ab Antibody
amp Ampicillin
AP Alkaline phosphatase
APS Ammoniumpersulfate
BCIP 5-bromo-4-chloro-3-indolyl-phosphate
BFA Brefeldin A
BiP Binding protein
bp Base pairs
cDNA Coding deoxyribonucleid acid
CT Carboxyterminus
D.m. Drosophila melanogaster
DMSO Diemethylsulfoxide
dNTP Deoxyribonucleoside Triphosphate
DOC sodium deoxycholate
DTT Dithiothreitol
e.g. exemplī grātia la(tin); for example
ECL Enhanced chemiluminescence
et al. et aliter
Fig Figure
fkh Forkhead
g Gravity
g Gram
h Hour/s
HRP Horseradish peroxidase
hs Heat shock
IF Immunofluorescence
IgG Immunoglobulin G
inx Innexin
IP immunoprecipitation
kD Kilo Dalton
l Liter
LB Luria-Bertani Medium
M Molaritymg Milligram
min Minute/s
ml Milliliter
mm Millimeter
mRNA Messenger ribonucleid acid
NaOH Natriumhydroxide
NBT Nito blue tetrazolium chloride
NEM N-ethylmaleimide
NT Aminoterminus
OD Optical density
PBS Phosphate buffered saline
PCR Polymerase-chain-reaction
PDI Protein disulfid isomerase
pH The negative of the logarithm to the base 10 of the concentration of hydrogen
ions
PMSF Phenylmethylsulfonyl fluoride
PVDF Polyvinylidene flouride
qRT-PCR Quantitative real time polymerase-chain-reaction
rpm Revolutions per minute
RT Room temperature
SDS Sodium dodecyl sulfate,
TAE Tris-acetate-EDTA
TE Tris-EDTA
TEMED N,N,N`, N`-tetramethylethylenediamine
TMA/DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate
U Unit
UV Ultraviolett
V Volt
v/v Volume per volume
w/v Weight per volume
WB Western Blot
Wt Wild type
µ Micro
µl MicroliterTable of Contents I
Table of Contents
1 Introduction
1.1 Mechanisms of cell-cell communicat.io.n....................................1.....................................
1.1.1 Cross talk between cell communication mechanisms ...........................3.........................
1.2 Epithelia and pola.r.it.y...................................................4....................................................
1.2.1 Epithelial junction.s. ...................................................4...................................................
1.2.2 Vertebrate gap junction life c.y.c.le.......................................8.........................................
1.3 Regulation of membrane trafficking in epithelial ..c.el.ls......................1.0.......................
1.3.1 Sorting of cargo in the trans-Golgi ne.t.w.o.r.k.............................1.0...............................
1.3.2 Apical transpo.r.t.....................................................1.1....................................................
1.3.3 Baso-lateral transport................................................1..2................................................
1.4 Actin and microtubule dependent transport of ve.s.ic.l.e.s....................1.3......................
1.4.1 Microtubule organisation.............................................................................................14
1.4.2 Microtubule based motors and transport of carg.o............................1.6.........................
1.4.3 Actin organisation.....................................................1.6..................................................
1.4.4 Actin based motors and transport of ca.rg.o................................1.7...............................
1.5 Objective of resear.c.h...................................................1.7.................................................
2 Material
2.1 Chemica.ls.............................................................1.8...........................................................
2.2 Expendable.s...........................................................1.8.........................................................
2.3 Equipme.n.t............................................................1.8..........................................................
2.4 Standards and K..it.s....................................................2.0...................................................
2.5 Antibodies..............................................................2.1..........................................................
2.5.1 Primary antibodie.s...................................................2.1..................................................
2.5.2 Secondary antibodies..................................................2.2...............................................
2.6 Solution.s..............................................................2.3...........................................................
2.7 Enzymes and Buff.e.r.s..................................................2.4.................................................
2.8 Plasmid.s..............................................................2.5............................................................
2.8.1 LD clo.n.e.s........................................................2.5........................................................
2.9 Oligonucleotide.s.......................................................2.6......................................................
2.9.1 Cloning prim.e.rs.....................................................2.6...................................................
2.9.2 Sequencing-prime.r.s .................................................2.7...............................................
2.9.3 q RT-PCR primer.s....................................................2.7..................................................Table of Contents II
2.10I n situ probes..........................................................2.8.......................................................
2.11 Organism.s............................................................2.8.........................................................
2.11.1 Bacterial strain.s.....................................................2.8..................................................
2.11.2 Fly str.ai.n.s.......................................................2.8.......................................................
2.11.3 Cell li.n.e.s........................................................2.9........................................................
2.12 Media for the cultivation of organ.is.m..s..................................3.0..................................
2.12.1 Media for bacterial culture.s............................................3.0.........................................
2.12.2 Media for fly cultivat.io.n..............................................3.0.............................................
3 Methods
3.1 Isolation of plasmids Efr.coomli .....................................................................................31
3.1.1 Analytical preparation..................................................3.1...............................................
3.1.2 Preparative scale .....................................................3.1...................................................
3.2 Isolation of RNA from tiss.u.e.s...........................................3.2...........................................
3.3 cDNA synthesis anqdR T-PCR set up..............................................................................32
3.3.1 cDNA synthe.si.s.....................................................32....................................................
3.3.2 qR T-PCR analysis...................................................