Biomedical Engineering Reference
In-Depth Information
against early endosome markers (e.g., EEA1) or late endosome
markers (e.g., CD63) can be used for colocalization studies
with the tagged EGF/EGFR complex.
7. View and image cells under the microscope. At different
chase times, ligand (EGF)- bound EGFRs (marked by
fl uorescence-labeled EGF) accumulate in membranous
endosomal structures with different morphologies, which
can be verifi ed based on size (
see
Fig.
1
) and specifi c marker
co-labeling (
see
Fig.
2
).
With the confocal microscope system, collect images in separate
z
sections. Final analysis of images could be from collapsed
z
stacks
or orthographic projections. For the LSM710 system and other
systems from the same vendor, image processing was done using
Zen 2010 software (Carl Zeiss):
3.2.2 Membrane Feature
and Colocalization
Analyses of Ligand-Bound
EGFR with Compartment
Markers
1. Size of membrane puncta can be measured using ImageJ (US
NIH, Bethesda, Maryland, USA), which enables the user to
perform particle size distribution analyses.
2. Specifi c marker co-labeling can be performed to identify the
membranous structures in which EGF-bound EGFR is found.
For example, the early endosomal marker EEA1 (early endo-
somal antigen 1) or late endosomal marker CD63/Lamp-3
can be used. Alternatively, other endosomal localized Rabs,
such as Rab11 in the recycling endosome, can also be used.
Visual inspection or calculation of overlap coeffi cient can be
performed using the Zen 2010 software to determine the per-
centage of EGF/EGFR membranous structures bearing the
various markers.
3.3 Biochemical
Analysis of Rab-EGFR
Complexes
To identify, confi rm, or study protein-protein interaction
between Rab proteins and their regulators and effectors, the
simplest and cheapest methods involve affi nity pulldown and
co-immunoprecipitation. Both methods are of low to moderate
sensitivity and rely on the interactions investigated being suffi -
ciently stable in cell lysates (
see
Note 7
). Co-immunoprecipitation,
in addition, relies on the availability of suitable antibodies.
Both methods could at best infer biochemical associations between
Fig. 1
(continued) of the different localizations of EGF/EGFR complex as it is traffi cked from cell surface (0 min)
to early (10 min) to late endosome (30 min). Colocalization between the ligand, EGF, and the receptor, EGFR, is
observed at all time points, indicating that EGFR remains ligand associated. Scale bar = 20
m. (
b
) Size of
puncta can be quantifi ed using ImageJ to determine the effect of various treatments on EGFR traffi cking. A431
cells transfected with scrambled (Scr) or Rab31 siRNA were pulsed with 0.5
ʼ
g/ml EGF-TxR and fi xed 30 min
after pulse. Size of EGF puncta was quantifi ed and presented here as a distribution of the number of puncta at
various sizes. Depletion of Rab31 shifted the distribution of puncta to smaller sizes, suggestive of inhibited
traffi cking to the late endosome
ʼ
