Cell-cell adhesion mediated by mobile receptor-ligand pairs [Elektronische Ressource] : a biomimetic study / vorgelegt von Susanne Franziska Fenz

Susanne Franziska Fenz

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 12 Mo

Extrait

Cell-cell adhesion mediated by mobile
receptor-ligand pairs: a biomimetic study
Dissertation
zur
Erlangung des Doktorgrades
der
Mathematisch-Naturwissenschaftlichen Fakult¨at
der
Rheinischen Friedrich-Wilhelms-Universit¨at Bonn
vorgelegt von
Susanne Franziska Fenz
aus
Frankfurt am Main
Bonn Dezember 2008Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakulta¨t
der Rheinischen Friedrich-Wihelms-Universit¨at Bonn
Erscheinungsjahr: 2009
1. Gutachter: Prof. Dr. Rudolf Merkel
2. Gutachter: Prof. Dr. Ulrich Kubitschek
Tag der Promotion: 20.03.09
DieseDissertationistaufdemHochschulschriftenserverderULBBonnunterhttp://hss.ulb.uni-
bonn.de/diss online elektronisch publiziert.meiner großartigen FamilieAcknowledgment - Vielen Dank ...
... meinem Doktorvater Herrn Prof. Rudolph Merkel fu¨r die M¨oglichkeit dieses
spannende Thema unter optimalen Bedingungen bearbeiten zu k¨onnen, fu¨r die
immerw¨ahrende Diskussionsbereitschaft, die bereitwilligen Erkl¨arungen und nicht
zuletzt die vermittelte Begeisterung fu¨r die Forschung an der Grenze von Physik,
Biologie und Chemie.
... my supervisor Kheya Sengupta for the introduction to the field of biomimetic
systems, for the great support from near and far, for your encouragement, patience
and hospitality!
... Ana Smith for your advice concerning the interpretation of the data from a theo-
retical point of view. Thank you very much for the last-minute reading of the thesis
and numerous skype sessions.
... Prof. Ulrich Kubitschek fu¨r die Zweitbetreuung dieser Arbeit. Vielen Dank fu¨r
die interessanten Gespr¨ache und Diskussionen in Bonn.
... Bernd Hoffman und Simone Born fu¨r die unermu¨dlichen Versuche dem E. Coli
ein leuchtendes E-cadherin abzuringen, fu¨r die kompetente und immer freundliche
Beratung in allen biologischen Fragen.
... Norbert Kirchgessner und Sebastian Houben fu¨r die Unterweisung in matlab, die
großeHilfsbereitschaftzuallenZeitenundUnzeitenunddieNot-Schokoladenrationen.
... CorneliaMonzelfu¨rdievielenaufschlussreichenDiskussionenunddasunermu¨dliche
Korrekturlesen! Ich danke Dirfu¨rDeine großeHilfsbereitschaft auch u¨berdas Fach-
liche hinaus.
... Sabine Dieluweit fu¨r die Versuche das E-cadherin mit dem AFM oder durch
Tieffrieren dingfest zu machen, außerdem fu¨r die immer bereitwillige und geduldige
chemische Beratung und nicht zuletzt fu¨r die Initative zu unseren sch¨onen Kultur-
ausflu¨gen.
... Christoph M¨ohl fu¨r die Einweisung in das vertrackte Confocal und die FRAP-
Technik.
... Agnes Csiszar fu¨r die Beratung in Lipid- und Vesikelfragen, fu¨r die nette ’Kel-
lergesellschaft’ und die lustigen - viel zu seltenen - Spieleabende.
... Dieter Waschbu¨sch fu¨r die biologische Beratung und die praktische Hilfe rund
ums Auto.... Claudia Cesa for you friendship, for the sleeping place in Ju¨lich, for the nice
girls-evenings and pancakes for breakfast.
... Wolfgang Rubner fu¨r die Beratung in allen technischen Fragen.
... dem Team der Werkstatt fu¨r die superschnelle Herstellung aller in dieser Arbeit
verwendeten Bauteile.
... Claudia Klamandt fu¨r die immer freundliche und hilfsbereite Unterstu¨tzung bei
verwaltungstechnischen Dingen.
... meiner Kochgruppe fu¨r das Durchfu¨ttern in den letzten Wochen!
... der gesamten Arbeitsgruppe des IBN-4 fu¨r die ganz besondere, freundschaftliche
Arbeitsatmosph¨are.
... Stefan, weil Du jeden Augenblick meines Lebens bereicherst. Dafu¨r, dass
Du mich in letzten Wochen ertragen hast, fu¨r die gemeinsam durchgearbeiteten
N¨achte, fu¨rs Haushalt schmeißen, fu¨rs Da-sein in jeder Zeit...
... meinerFamiliefu¨rdieliebevolleUnterstu¨tzung,fu¨rdieunz¨ahligenBriefe,Carepakete,
Besuche und Anrufe, fu¨r die unermu¨dliche Aufmunterung, Motivation und Liebe!Abstract
Adhesion of cells to other cells is of vital importance for multicellular organisms.
It is mediated by specific bonds between cell surface molecules. These molecules
can be either attached to some rigid internal structure of the cell or freely diffus-
ing within the membrane. The present work aims at a quantitative understanding
of the physical processes in cell-cell adhesion caused by such mobile cell adhesion
molecules.
To unequivocally separate physical processes from active biological cell response,
a biomimetic model system was developed. It consisted of a giant unilamellar
vesicle (GUV)adhering via specific ligand-receptor interactions toa solid supported
lipid bilayer (SLB). Adhesion was mediated by either biotin-neutravidin (an avidin
analogue) or the extracellular domains of the homophilic cell adhesion molecule E-
cadherin (Ecad). While the biotin-neutravidin interaction is known to be extremely
strong (Gibbs free energy of bonding ΔG in solution ∼ 35 k T) the Ecad-Ecad0 B
bond represented the case of very weak binding (ΔG ∼ 2 k T). In each case, re-0 B
ceptors and ligands were bound to the SLB and the membrane of the GUV where
they were able to freely diffuse laterally.
Microinterferometry (reflection interference contrast microscopy, RICM) was em-
ployed to dynamically quantify vesicle shapes with lateral resolution of ∼ 250 nm
and axial resolution of a few nanometers. Fluorescence microscopy (FM) yielded
lateral distributions and diffusivities of fluorescently labelled membrane molecules.
A microscope was modified for simultaneous application of FM and RICM. In ad-
dition, the quantitative interpretation of RICM images was extended.
In membrane adhesion mediated by both binding pairs in high concentration the
final equilibrium was characterized with respect to membrane distance, tension,
and adhesion energy density with the help of RICM. The adhesion energy densities
andtensions inthestrongbindingcase werebeyond themeasurable rangeaccessible
−6 2 −5 2with the applied technique (>10 J/m and >10 N/m ) while for weak binding
−7 2 −6 2considerably lower values were measured (10 J/m and 10 N/m ). The measuredinter-membrane distance of 7 nm for biotinylated vesicles adhered via neutravidin
was in very goodagreement with the expected height calculated from the molecular
size of neutravidin plus the lengths of the biotin linkers. The corresponding mea-
surement of 50 nm on the Ecad bound vesicle indicates that here Ecad was bound
by only partial overlap of its extracellular domains.
The fluidity of the SLB and the diffusion of the receptors was studied during
vesicle adhesion. Continuous photobleaching (CP)was used tomeasure the in-plane
diffusion of tracer lipids and receptors in the SLB membrane. In the strong inter-
action (biotin-neutravidin) case, binding of soluble receptors to the SLB alone led
to reduced diffusion of tracer lipids. From theoretical considerations, this could be
attributedpartiallytointroductionofobstaclesandpartiallytoviscous effects. Fur-
ther specific binding of a GUV membrane caused additional slowing down of tracers
(up to 15%) and immobilization of receptors, and led to accumulation of receptors
in the adhesion zone till full coverage was achieved. It was shown that a crowding
effect due to the accumulated receptors alone was not sufficient to account for the
slowing down - an additional friction from the membrane also played a role. In the
weak binding case (Ecad), no significant change in diffusion of tracer lipids was ob-
served upon protein binding and subsequent vesicle binding. Here, the insensitivity
of diffusion to membrane binding arose due to the large inter-membrane distance
which reduced membrane induced friction. It was concluded that the effect of inter-
membrane adhesion on diffusion depended strongly on the choice of the receptors.
In the strong binding case vesicle adhesion was achieved at low and high concen-
trations of receptors and ligands while the weak binding case required high concen-
trations. As a consequence, the dynamics of vesicle adhesion were studied for
a broad range of receptor-ligand concentrations for strong binding and merely at
high concentrations for weak binding. It was shown that low ligand concentrations
in the vesicle retarded the establishment of the initial nucleation center while low
concentrations of receptors on the bilayer mainly influenced the final adhered state.
Complete adhesion and full receptor accumulation was reached only above a thresh-
old concentration. Simultaneous observation of the adhered area in RICM and the
corresponding receptor distribution influorescence revealed thatadhesion andaccu-
mulation went hand in hand above the threshold but atlower concentrations a large