Nanoscale imaging of restricted cell membrane receptor diffusion [Elektronische Ressource] / von Christian Tischer

Christian Tischer

Sujets

• Physik
• Biologie

Informations

• Publié par: charite_-_universitatsmedizin_berlin
• Publié le: 01 janvier 2005
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 28 Mo

Extrait

Nanoscale imaging of restricted cell membrane receptor
di usion
DISSERTATION
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biophysik
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultat I
Humboldt-Universitat zu Berlin
von
Herr Dipl.-Phys. ChristianTischer
geboren am 13.12.1973 in Stuttgart
Prasident der Humboldt-Universitat zu Berlin:
Prof. Dr. Jurgen Mlynek
Dekan der Mathematisch-Naturwissenschaftlichen Fakultat I:
Prof. Thomas Buckhout, PhD
Gutachter:
1. Prof. Dr. Reinhart Heinrich
2. Prof. Dr. Philippe Bastiaens
3. Prof. Dr. Ernst-Ludwig Florin
Tag der mundlichen Prufung: 9. Juni 2005iiAbstract
Intheworkpresented,anovelimagingtechnique(TNIM-ThermalNoiseImagingMicroscopy)
was developed for the purpose of studying nanoscale di usive motion in heterogeneous me-
dia. TNI-Microscopy was specically used to investigate if the mobility of cell membrane
receptors is inuenced by lateral membrane nanostructures. The direct investigation of these
two-dimensional ultrastructures in living cells was up to now hampered because of lacking
microscopy techniques. In TNI-Microscopy, the di usive motion of a nanoparticle is limited
to a small volume, making it possible to observe constraints on the particle’s motion. Thus,
the particle can be used to sample nanostructures. Concomitant, it is also recorded how the
dynamics of the particle’s motion are inuenced by hydrodynamic coupling to the sampled
structures. To realise TNI-Microscopy, a microscope was built that employs an optical trap
to limit the di usive motion of a probe particle to a submicroscopic volume. Within this
volume, the particle position uctuations are tracked with nanometer spatial precision and
microsecond temporal resolution using an interferometric position detector. Initially, experi-
ments on rigid three-dimensional structures were conducted, demonstrating that objects such
as the laments of a polymer network can be clearly seen in histograms of the particle’s po-
sition uctuations. From these histograms the position of objects could be determined with
a precision of about 10 nm in three dimensions. These experiments were of fundamental sig-
nicance as they conrmed that di usive motion of a probe particle can indeed be used to
image nanoscopic objects. In subsequent experiments it could be shown that it is simultane-
ously possible to capture the dynamics of the three-dimensional particle position uctuations.
Existing hydrodynamic theories that describe the mobility of a sphere (probe particle) at
di erent distances to a rigid interface could be validated from the nanometer to the microm-
eter scale. Thus, the fundamental concepts of TNI-Microscopy were established and could be
used to study how the mobility of cell membrane receptors is inuenced by membrane lateral
nanostructures. To achieve this, nanoparticles were bound specically to the cell membrane
epidermal growth factor (EGF) receptor. In this case, the particle served to make the lateral
motionoftheEGF-receptorsobservableandtolimititsmotiontoasubmicroscopicmembrane
area. Thus, the motion of EGF-receptors could be observed in a small membrane area for any
length of time. In strong contrast to the measurements on rigid objects, the lateral membrane
structure appeared highly dynamic. Inaccessible nanoscopic membrane areas restricted the
di usivemotionofthereceptor. Thecomparisonwithalipidanchoredproteinshowedthatthe
characteristics of these areas depended on the observed protein. Furthermore, it was possible
to observe these areas changing shape and position on the second time scale. A consequence
iiiof the restriction by these areas was that the EGF-receptor dwelt up to 20 times longer in
specic membrane regions as it would have been expected for free di usion. Moreover, it was
possible to obtain information on the dynamics of the receptor’s motion on the nanometer
and the micrometer scale. The analysis of these dynamics provided evidence that the di u-
sion of the receptor is dominated by the lipid bilayer structure of the cell membrane on short
length scales, whereas the motion on long length scales is hindered by nanoscopic membrane
heterogeneities. In addition,