Sites of Ca2+ wave initiation move with caveolae to the trailing edge of migrating cells
Masashi Isshiki1,
Joji Ando2,
Kimiko Yamamoto2,
Toshiro Fujita3,
Yunshu Ying1 and
Richard G. W. Anderson1,*
1 Department of Cell Biology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235-9039, USA
2 Department of Biomedical Engineering, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo113-8655, Japan
3 Department of Nephrology and Endocrinology, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo113-8655, Japan
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Fig. 4. Quantification of caveolin-1 distribution in shear stressed cells.
Endothelial cells were either exposed to the indicated laminar shear stress
force for 24 hours (right panel) or to a constant force of 20
dynes/cm2 for different times (left panel). They were then fixed
and processed for localization of caveolin-1. Representative cells were picked
and scored according to whether the caveolin-1 staining was principally in one
of five regions of the cell (top left), designated A, B, C, D or E. Region A
corresponded to the most upstream region of the cell. The percent of cells in
each group (ordinate) as a function of time or force (abscissa) was then
plotted.
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Fig. 1. Polarization of caveolin-1 during cell migration. Cell migration was
induced by scraping the cells from one half of the coverslip. Primary
endothelial cells were grown to confluency on glass coverslips as described.
On day zero, one half of the cells on the coverslip were removed by scraping
(below the yellow line) and the remaining cells were either processed directly
(0 hr) for indirect immunofluorescence staining with the indicated antibody or
allowed to grow for 4 and 24 hours before processing. The distribution of
caveolin (left) and actin (right) are shown in the same cell. Yellow arrows
point to regions high in caveolin-1 staining in migrating cells. Bar, 100
µm.
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Fig. 2. Polarization of caveolin-1 in response to fluid shear stress. Primary
endothelial cells were either cultured on coverslips (unstressed) or exposed
to a fluid shear stress at a force of 20 dynes/cm2 (stressed) in a
parallel-plate flow chamber for 24 hours as described. Cells were then
processed for colocalization of the indicated protein by indirect
immunofluorescence. White arrows indicate regions in stressed cells that were
rich in caveolin-1 staining. These arrows also point in the direction of fluid
flow. The yellow asterisk marks a cell extension that is rich in caveolin-1.
Bar, 20 µm.
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Fig. 3. Fluid shear stress does not cause polarization of clathrin AP-1/2 or
microtubules. Endothelial cells were exposed to laminar shear stress as
described in the legend to Fig.
2. Cells were then fixed and processed to localize the indicated
protein. Arrows indicate regions where caveolin-1 has accumulated and are
pointing in the direction of fluid flow. Bar, 20 µm.
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Fig. 5. Caveolae are concentrated in the upstream region of shear stressed cells.
Primary endothelial cell cultures were grown on plastic coverslips instead of
glass coverslips and exposed to 20 dynes/cm2 shear stress for 24
hours. The coverslips were marked to indicate the direction of laminar flow,
fixed in glutaraldehyde and oriented in the Epon plastic during embedding so
that sections could be made perpendicular to plane of the coverslip. Thin
sections were made and viewed directly. Large arrow indicates the direction of
laminar flow while the small arrows (inset) indicate regions where caveolae
have accumulated. The white asterisk is a region where caveolae appear to be
interacting with smooth ER. Bar, 0.2 µm (A); 0.1 µm (B).
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Fig. 6. Caveolin-1 mRNA and protein do not change in stressed cells. Endothelial
cells were exposed to a shear stress of 20 dynes/cm2 for 1, 3, 6,
12 or 24 hours. Cells were then processed either for immunoblotting of
caveolin-1 (A) or RT/PCR analysis of caveolin mRNA (B) as described. The
appropriate band for caveolin-1 is indicated in each gel. The mRNA for GAPDH
(3-phosphate glyceraldehyde dehydrogenase) was used as a load control.
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Fig. 7. Simultaneous relocation of caveolin-1 and G q/11 in
response to shear stress. Endothelial cells were exposed to a shear stress of
20 dynes/cm2 for 24 hours. Endothelial cells were exposed to
laminar shear stress (arrow indicates direction of flow) as described in the
legend to Fig. 2. Cells were
then fixed and processed to localize the indicated protein. Bar, 30 µm.
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Fig. 8. Sites of Ca2+ wave initiation in unstressed (A-D) and stressed
(E-H) cells. Primary endothelial cells were either cultured on coverslips
(unstressed) or exposed to a fluid shear stress of 20 dynes/cm2
(stressed) from the right to the left for 24 hours in a parallelplate flow
chamber. Both sets of cells were loaded with the Ca2+ sensing dye
Indo-1 (5 µM) before incubating the cells in the presence of either 0.5
µM ATP (unstressed cells) or 2 µM ATP (stressed cells). Images were
taken at 0.38 second intervals of a representative cell to visualize
Ca2+ release. At the end of the recording, the coverslip was fixed
and processed to localize caveolin-1 and actin. Cell morphology was used to
match Ca2+ release with caveolin-1 and actin staining. Bar, 20
µm.
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Fig. 9. Shear stress changes the sensitivity of cells to ATP. Primary endothelial
cells were either cultured on coverslips (unstressed) or exposed to a fluid
shear stress of 20 dynes/cm2 (stressed) for 24 hours in a
parallel-plate flow chamber. The cells were loaded with Indo-1 and then
exposed to the indicated concentrations of ATP while Ca2+-dependent
Indo-1 fluorescence was continuously recorded. As little as 0.2 µM ATP was
sufficient to stimulate a wave of Ca2+ release in unstressed cells,
whereas 2 µM ATP was required to elicit a similar response in stressed
cells.
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© The Company of Biologists Ltd 2002
