Chemistry Reference
In-Depth Information
6
Dynamics of Membrane Receptors: Single-molecule Tracking
of Quantum Dot Liganded Epidermal Growth Factor
Guy M. Hagen, Keith A. Lidke, Bernd Rieger, Diane S. Lidke, Wouter Caarls,
Donna J. Arndt-Jovin, and Thomas M. Jovin
6.1
Introduction
The erbB family of receptor tyrosine kinases (RTKs) includes erbB1 (the classical
epidermal growth factor (EGF) receptor, hereafter referred to as EGFR), erbB2, erbB3
and erbB4. Activation of these transmembrane proteins initiates signaling cascades
controlling numerous cellular processes such as DNA replication and division.
Binding of speci
c peptide ligands to the ectodomains of the RTKs leads to auto- and
transactivation of the cytoplasmic protein kinase domains. The activated receptors
bind adaptor proteins, initiating several signal transduction cascades, such as those
mediated by MAP kinases. The fate of the activated receptors is complex: endocytosis
via coated pits, covalent modi
cation (deactivation by enzymatic dephosphorylation
and ubiquitinylation), and endosomal traf
cking leading to proteosomal and/or
lysosomal degradation or recycling to the plasmamembrane. The overexpression and
unrestrained activation of the erbB family are implicated in many types of cancer [1].
We have shown in previous publications [2, 3] that quantum dots (QDs) bearing
natural ligands function as effector molecules and provide the means for prolonged
real-time visualizations of erbB molecules on living cells. The multiple steps of the
signaling pathways can be followed, and detailed movies of image sequences of the
underlying mole-cular processes can be generated (available as supplementary
information in [2, 3]).
For the studies reported here, biotinylated EGF was bound to commercial
streptavidin-conjugated QDs. QDs have unique features providing many advantages
for cellular imaging: (i) high absorption cross-sections and quantum yields, permit-
ting detection down to the single nanoparticle level and reliable quantitation of
binding and transport phenomena; (ii) extreme photostability, allowing imaging
over prolonged periods; (iii) a broad excitation spectrum rising toward the UV,
allowing the simultaneous excitation of visible
fluorescent proteins (VFP) and QDs;
and (iv) narrow emission bands across the visible spectrum. QDs can be regarded as
single molecule probes [4].
