Biomedical Engineering Reference
In-Depth Information
of candidate proteins in transport by gene silencing or expression
of mutant proteins, quantitative assays are required. Chemically
modifi ed and/or radioactive ligands or surface-labeled receptors
may be followed biochemically by monitoring the disappearance
or reappearance of surface accessibility. Fluorescent molecules
may be followed microscopically with the added benefi t of mor-
phological information.
A particularly useful system to analyze receptor endocytosis
and recycling is the transferrin (Tf) receptor, the prototype of a
transport receptor cycling between the plasma membrane and
endosomes. Tf acts as the transporter of Fe
3+
ions in the blood-
stream. Loaded holo-Tf binds to the Tf receptor at neutral pH and
is rapidly endocytosed at rates of ~15 %/min. In the more acidic
environment of early endosomes, the iron is released, while apo-Tf
remains bound to the receptor, which is recycled to the plasma
membrane either directly from early sorting endosomes or via
recycling endosomes. At neutral extracellular pH, apo-Tf is released
from the receptor [
5
,
7
,
8
]. At steady state, approximately 80 % of
the Tf receptor is intracellular, while 20 % is at the plasma mem-
brane [
9
-
11
].
To follow traffi cking of Tf, it needs to be labeled. Iodination
with radioactive [
125
I] iodine provides high sensitivity. Biotinylated
Tf (which is also commercially available) allows easy detection by
streptavidin-horseradish peroxidase blotting after SDS-gel elec-
trophoresis or directly on the culture dish with a microplate reader.
A further possibility is the use of fl uorescent Tf to measure the
fl uorescence intensity of either entire cell populations or single
cells using fl ow cytometry or fl uorescence microscopy. To study
endocytosis, cells are incubated with fl uorescent Tf, and endocy-
tosis is measured as the increase of intracellular fl uorescence over
time. To investigate recycling from endosomes back to the plasma
membrane, cells are fi rst loaded with fl uorescent Tf and subse-
quently the surface-bound Tf is removed. The cells are then incu-
bated at 37 °C to allow recycling of Tf in the presence of an iron
chelator to prevent rebinding of the recycled Tf. The time course
of release of fl uorescent Tf is monitored.
Here, we describe the use of fl uorescently labeled Tf and fl uo-
rescence microscopy for quantitative endocytosis and recycling
assays using automated fl uorescence imaging and image analysis
(Fig.
1
). Using this automated analysis process, it is possible to
measure the fl uorescence intensity of Tf in thousands of individual
cells at different time points. It is further possible to measure Tf in
transiently transfected cell populations distinguishing transfected
from untransfected cells, high expressers from low expressers, or
large cells from small ones. The simultaneous measurement of two
conditions in the same experiment provides additional confi dence
in the signifi cance of observed rate differences.
