doi: 10.1242/10.1242/jcs.00177
SARA, a FYVE domain protein, affects Rab5-mediated endocytosis
Yang Hu1,
Jen-Zen Chuang2,
Kai Xu1,
Timothy G. McGraw3 and
Ching-Hwa Sung2,4,*
1 Graduate Program of Neuroscience, Weill Medical College of Cornell University,
1300 York Avenue, New York, NY 10021, USA
2 Department of Ophthalmology, Weill Medical College of Cornell University, 1300
York Avenue, New York, NY 10021, USA
3 Department of Biochemistry, Weill Medical College of Cornell University, 1300
York Avenue, New York, NY 10021, USA
4 Department of Cell and Development Biology, Weill Medical College of Cornell
University, 1300 York Avenue, New York, NY 10021, USA
View larger version (111K):
[in a new window]
|
Fig. 1. Localization of endogenous SARA on early endosomal membranes. Low
magnification (A-C, Bar, 10 µm) and 4 selective views of high magnification
(D-F, Bar, 2 µm) images obtained from a single confocal section (0.6 µm)
of HEK cells double labeled for SARA (A,D) and EEA1 (B,E). The images reveal
heterogeneous pools of early endosomes displaying various levels of SARA and
EEA1. Some early endosomes have a higher level of SARA, while others have a
higher level of EEA1. SARA and EEA1 did not always overlap on the membranes of
individual early endosomes.
|
|
View larger version (86K):
[in a new window]
|
Fig. 2. Reversal of SARA-mediated endosome enlargement by Rab5S34N overexpression.
MDCK cells cotransfected with GFP-SARA (green) with Rab5Q79L (A-C) or Rab5S34N
(D-F) were fixed for Rab5 immunostaining (red). Rab5 transfected cells can be
easily distinguished from the neighboring untransfected cells based on their
higher level of Rab5 immunolabeling. Because the photographic exposure was
chosen to highlight the transfected cells, the endogenous Rab5 labeling is
negligible in these figures. (A-C) Rab5Q79L labeling is extensively
colocalized with SARA on enlarged early endosomal membranes. (D-F) In contrast
to the cells singly transfected with GFP-SARA, in which green fluorescence is
observed on enlarged endosomal membranes (arrowheads), GFP signals are
distributed diffusely in the cytosol and on small puncta in the cells
co-transfected with GFA-SARA and Rab5S34N (arrow). Bar, 10 µm.
|
|
View larger version (67K):
[in a new window]
|
Fig. 3. Effect of SARA on Tf endocytosis. (A) GFP-SARA (green)-transfected HEK
cells were loaded with Cy3-Tf (red) at 37°C for 6, 15 or 120 minutes
before the cells were chilled, washed and fixed for GFP and Cy3 visualization.
At 6 minutes, Cy3-Tf was readily detectable on small endosomes in the
untransfected control cells, but not in the GFP-SARA transfected cells
(arrows). At 15 minutes, Cy3-Tf becomes detectable in the GFP-SARA transfected
cells. After the 2-hour loading, Cy3-Tf reached a steady-state occupancy and
was distributed on both early and recycling endosomes in the control cells,
whereas Cy3-Tf was largely concentrated in the SARA-positive compartments of
the transfected cells. Bar, 10 µm. (B) HEK cells cotransfected with
GFP-SARA and Rab5S34N were loaded with Cy3-Tf for 6 minutes before fixation.
The level of internalized Cy3-Tf is indistinguishable between the
double-transfected cells (indicated by cytosolic GFP-SARA signal) and the
neighboring untransfected cells. (C) SARA stable transfectants and HEK cells
grown in 6-well dishes were incubated with 125I-Tf at 37°C for
the time indicated. The amounts of total, internalized and surface-associated
radioactivity were determined. Data are plotted as described in Wiley and
Cunningham (Wiley and Cunningham,
1982 ), such that the slope is equal to the endocytic rate
constant, Ki. Results shown are from a single experiment
and are representative of those obtained on three separate occasions. (D)
SARA-transfected cells and HEK cells grown on coverslips were loaded for 2
hours with Cy3-Tf in serum-free medium. The cells were then fixed and the
surface TfR was labeled with Cy5 by indirect immunofluorescence. Quantitative
data was obtained by summing the Cy3 and Cy5 fluorescence in each field
(>20 cells per field), taking the ratio and averaging over multiple fields.
The ratio of Cy5/Cy3 fluorescence, the index of the surface/total TfR, was
obtained from the average of three independent experiments
(means±s.e.m.).
|
|
View larger version (83K):
[in a new window]
|
Fig. 4. The effect of overexpression of SARA and Rab5 mutants on surface TfR
distribution. HEK cells transfected with GFP-SARA (A,B), Rab5Q79L (C,D),
Rab5S34N (E,F) and both GFP-SARA and Rab5S34N (G,H) were fixed. Without
permeabilization, the cells were labeled for surface TfR using an antibody
recognizing the extracellular domain of TfR (B3/25), followed by Alexa594
anti-mouse IgG (B,D,F,H). Cells overexpressing GFP-SARA and Rab5Q79L, but not
Rab5S34N, appear to have reduced surface TfR labeling (*). In
contrast, cells double-transfected with GFP-SARA and Rab5S34N have similar
levels of surface TfR compared with the neighboring cells (*). Bar,
10 µm.
|
|
View larger version (73K):
[in a new window]
|
Fig. 5. Reduced Tf recycling in cells overexpressing SARA. (A) HEK cells
transiently transfected with GFP-SARA were loaded with Cy3-Tf at 37°C for
2 hours and chased in the presence of excess unlabeled Tf and desferroxamine
for 0, 10, and 30 minutes before the cells were chilled, fixed and observed.
At all time points, Cy3-Tf was mainly accumulated in the GFP-SARA positive
endosomal compartments (arrows). (B) HEK cells double-transfected with
GFP-SARA and Rab5S34N were preloaded with Cy3-Tf for 2 hours and chased for 30
minutes. The double transfected cells (indicated by cytosolic GFP signal)
release Cy3-Tf in a manner indistinguishable from the neighboring cells. Bar,
10 µm. (C) The 125I-labeled Tf was loaded on HEK stably
expressed SARA or HEK cells for 2 hours before the chase. The natural
logarithm of the percent of Tf remaining inside the cell versus the chase time
from one experiment is shown. (D) The mean±s.e.m. of the recycling rate
constants from three independent experiments are presented. The Tf recycling
rate constant of the SARA stable line is significantly slower than that of HEK
control cells (P<0.01, two-tailed Student's t test).
|
|
© The Company of Biologists Ltd 2002
