Please summarize these two papers and explain what the figure tells us. Elaborate the details as if your explaining the whole class what this paper is about. Please give a clear understanding of what each of the figures tells us and thier meanings.
Please summarize these two papers and explain what the figure tells us. Elaborate the details as if your explaining the whole class what this paper is about. Please give a clear understanding of what each of the figures tells us and thier meanings.
Biology 2e
2nd Edition
ISBN:9781947172517
Author:Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:Matthew Douglas, Jung Choi, Mary Ann Clark
Chapter9: Cell Communication
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Please summarize these two papers and explain what the figure tells us. Elaborate the details as if your explaining the whole class what this paper is about. Please give a clear understanding of what each of the figures tells us and thier meanings.
![a
Before
b
[Ca²+], (a.u.)
C
Before
2.5
After
2.0
CytD
1.5
1.0
Percent increase (%)
0
20
15
10
5
0
CytD
Na,K-ATPase and InsP3 Receptor in a Signaling Microdomain
After
CytD
Ouabain
5 10 15 20 25 30
Time (min)
FRET
*
IP
NKAa1 InsP₂R2 InsP,R3
WB: InsP₂R3
FIG. 4. Effect of cytoskeleton perturbation on physical associ-
ation between Na,K-ATPase and InsP.R. In a, the actin cytoskele-
ton was disrupted after CytD (5 μM) treatment in GFP-actin-expressing
RPT cells. In b, CytD abolished ouabain-induced Ca²+ oscillations in
RPT cells. Arbitrary units (a.u.) represent ratio values corresponding to
intracellular Ca2+ concentration changes. c and d, FRET measure-
ments between Na,K-ATPase and InsP3R3. c, GFP-NKAal images of
COS-7 cells with and without CytD treatment before and after acceptor
photobleaching (bleached area indicated by square). d, quantitative
changes in emission intensities after bleaching as compared with before
bleaching, mean ± S.E., *, p < 0.05. FRET was eliminated by CytD. e,
co-immunoprecipitation (IP) studies followed by Western blotting (WB)
for InsP3R3 in CytD-treated COS-7 cells. InsP³R3 did not co-immuno-
precipitate with Na,K-ATPase.
50359
central role for induction of ouabain-induced Ca²+ oscillations.
We also performed a GST pull-down assay where GST was
fused to the full-length (95 amino acids) NH₂ terminus of the
Na,K-ATPase al-subunit. GST-NKAa1.N95 pulled down
InsP3R3 from a lysate of RPT cells, whereas GST alone did not
pull down InsP3R3 (Fig. 5e).
It was demonstrated previously that NF-kB, a well known
Ca²+-dependent transcription factor, is more readily activated
by low frequency Ca²+ oscillations than by a sustained Ca²+
increase (30). To exploit this effect and to determine the down-
stream functional implications of disturbing the communica-
tion between the NH₂ terminus of Na,K-ATPase al-subunit
and InsP3R, we compared NF-kB responsiveness to ouabain in
cells expressing GFP-NKAa1.M32 with neighboring cells that
only expressed the endogenous Na,K-ATPase al-subunit. Fig.
6a shows a GFP-NKAa1.M32-expressing cell, and Fig. 6b
shows NF-kB staining of this cell and its neighboring, untrans-
fected cells in the same field of view following ouabain treat-
ment for 30 min. NF-kB activation was semiquantitatively
estimated by measuring the ratio of NF-kB nuclear signal to
cytosolic signal for each cell in the same field of view. Results
from this analysis indicated that ouabain caused nuclear trans-
location of NF-kB in non-transfected cells but was without
effect in GFP-NKAa1.M32-expressing cells (Fig. 6c). These
findings demonstrate that truncation of the NH₂ terminus of
Na,K-ATPase al-subunit results in a functional Na,K-ATPase
that resides at the plasma membrane yet is sufficient to disrupt
ouabain-induced activation of NF-kB.
DISCUSSION
It is now generally agreed that many, if not most, important
processes in the cell are controlled by proteins aggregated
together in complexes (21, 22, 31-33). The assembly of com-
plexes that contain a receptor and components of signal ma-
chinery provides the cell with a highly selective means to
engage a specific signaling pathway. The finding that ligand-
bound Na,K-ATPase assembles with InsP3R and that this as-
sembly can give rise to intracellular Ca²+ oscillations with a
constant periodicity in the minute range represents a novel
principle for such a protein complex.
Based on our combined results, we suggest that the ouabain-
induced Ca²+ oscillation and signal-transducing function of
Na,K-ATPase is made possible by the local organization of
Na,K-ATPase and InsP3R into a spatially organized functional
microdomain that links the plasma membrane to intracellular
ER Ca²+ stores. A signaling microdomain can function without
a diffusible messenger provided that the transducer and the
effector are in such proximity that they can communicate via
protein-protein interaction, either directly or via one or more
interacting/scaffolding proteins. Molecular strategies using the
InsP3 sponge and pharmacological studies using the PLC in-
hibitor, U73122, indicate that the ouabain-induced Ca²+ oscil-
latory response may be elicited via an InsP-independent
mechanism of InsP3R activation. Significant energy transfer
between the donor and acceptor complexes, GFP-NKAa1 and
anti-InsP3R-anti-mouse IgG-Cy3, indicate a distance of less
than 12 nm (16). It is well established that the ER is juxtaposed
to the plasma membrane (34). FRET was recorded in a region
of the plasma membrane, and the result is clearly compatible
with previous electron microscopy studies showing that the
distance between the plasma membrane, where Na,K-ATPase
is located, and the membrane of ER, where the InsP3R is
located, can be as short as 10 nm (35). It should be noted,
however, that FRET is recorded between the GFP-labeled
Na,K-ATPase and Cy3-labeled goat-anti-mouse IgG antibody
that binds to the InsP3R antibody. Taking the size of the
antibodies and the GFP molecule into account, the maximal](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F10b2558f-14bb-4a79-9eba-8554e0210a1c%2F32af8b42-0a77-475f-bd40-dc10e67a553e%2Fdre1dk_processed.jpeg&w=3840&q=75)
Transcribed Image Text:a
Before
b
[Ca²+], (a.u.)
C
Before
2.5
After
2.0
CytD
1.5
1.0
Percent increase (%)
0
20
15
10
5
0
CytD
Na,K-ATPase and InsP3 Receptor in a Signaling Microdomain
After
CytD
Ouabain
5 10 15 20 25 30
Time (min)
FRET
*
IP
NKAa1 InsP₂R2 InsP,R3
WB: InsP₂R3
FIG. 4. Effect of cytoskeleton perturbation on physical associ-
ation between Na,K-ATPase and InsP.R. In a, the actin cytoskele-
ton was disrupted after CytD (5 μM) treatment in GFP-actin-expressing
RPT cells. In b, CytD abolished ouabain-induced Ca²+ oscillations in
RPT cells. Arbitrary units (a.u.) represent ratio values corresponding to
intracellular Ca2+ concentration changes. c and d, FRET measure-
ments between Na,K-ATPase and InsP3R3. c, GFP-NKAal images of
COS-7 cells with and without CytD treatment before and after acceptor
photobleaching (bleached area indicated by square). d, quantitative
changes in emission intensities after bleaching as compared with before
bleaching, mean ± S.E., *, p < 0.05. FRET was eliminated by CytD. e,
co-immunoprecipitation (IP) studies followed by Western blotting (WB)
for InsP3R3 in CytD-treated COS-7 cells. InsP³R3 did not co-immuno-
precipitate with Na,K-ATPase.
50359
central role for induction of ouabain-induced Ca²+ oscillations.
We also performed a GST pull-down assay where GST was
fused to the full-length (95 amino acids) NH₂ terminus of the
Na,K-ATPase al-subunit. GST-NKAa1.N95 pulled down
InsP3R3 from a lysate of RPT cells, whereas GST alone did not
pull down InsP3R3 (Fig. 5e).
It was demonstrated previously that NF-kB, a well known
Ca²+-dependent transcription factor, is more readily activated
by low frequency Ca²+ oscillations than by a sustained Ca²+
increase (30). To exploit this effect and to determine the down-
stream functional implications of disturbing the communica-
tion between the NH₂ terminus of Na,K-ATPase al-subunit
and InsP3R, we compared NF-kB responsiveness to ouabain in
cells expressing GFP-NKAa1.M32 with neighboring cells that
only expressed the endogenous Na,K-ATPase al-subunit. Fig.
6a shows a GFP-NKAa1.M32-expressing cell, and Fig. 6b
shows NF-kB staining of this cell and its neighboring, untrans-
fected cells in the same field of view following ouabain treat-
ment for 30 min. NF-kB activation was semiquantitatively
estimated by measuring the ratio of NF-kB nuclear signal to
cytosolic signal for each cell in the same field of view. Results
from this analysis indicated that ouabain caused nuclear trans-
location of NF-kB in non-transfected cells but was without
effect in GFP-NKAa1.M32-expressing cells (Fig. 6c). These
findings demonstrate that truncation of the NH₂ terminus of
Na,K-ATPase al-subunit results in a functional Na,K-ATPase
that resides at the plasma membrane yet is sufficient to disrupt
ouabain-induced activation of NF-kB.
DISCUSSION
It is now generally agreed that many, if not most, important
processes in the cell are controlled by proteins aggregated
together in complexes (21, 22, 31-33). The assembly of com-
plexes that contain a receptor and components of signal ma-
chinery provides the cell with a highly selective means to
engage a specific signaling pathway. The finding that ligand-
bound Na,K-ATPase assembles with InsP3R and that this as-
sembly can give rise to intracellular Ca²+ oscillations with a
constant periodicity in the minute range represents a novel
principle for such a protein complex.
Based on our combined results, we suggest that the ouabain-
induced Ca²+ oscillation and signal-transducing function of
Na,K-ATPase is made possible by the local organization of
Na,K-ATPase and InsP3R into a spatially organized functional
microdomain that links the plasma membrane to intracellular
ER Ca²+ stores. A signaling microdomain can function without
a diffusible messenger provided that the transducer and the
effector are in such proximity that they can communicate via
protein-protein interaction, either directly or via one or more
interacting/scaffolding proteins. Molecular strategies using the
InsP3 sponge and pharmacological studies using the PLC in-
hibitor, U73122, indicate that the ouabain-induced Ca²+ oscil-
latory response may be elicited via an InsP-independent
mechanism of InsP3R activation. Significant energy transfer
between the donor and acceptor complexes, GFP-NKAa1 and
anti-InsP3R-anti-mouse IgG-Cy3, indicate a distance of less
than 12 nm (16). It is well established that the ER is juxtaposed
to the plasma membrane (34). FRET was recorded in a region
of the plasma membrane, and the result is clearly compatible
with previous electron microscopy studies showing that the
distance between the plasma membrane, where Na,K-ATPase
is located, and the membrane of ER, where the InsP3R is
located, can be as short as 10 nm (35). It should be noted,
however, that FRET is recorded between the GFP-labeled
Na,K-ATPase and Cy3-labeled goat-anti-mouse IgG antibody
that binds to the InsP3R antibody. Taking the size of the
antibodies and the GFP molecule into account, the maximal
![50360
NKAa1 (MGKGV)
NKAa1.M32
b
GFP-NKAa1.M32
Na,K-ATPase and InsP3 Receptor in a Signaling Microdomain
30
GRDKYEPAAVSEHGDKKSKKAKKERDMDELKKEVSMDDHKLSLDELHRKYGTDLSRGLT PARAAEILARDGPNALTPPPTTPEWVKFCRQ...
a
1
[Na], (a.u.)
1.6
1.4
1.2
1.0
10
-10
20
0
10 20
Time (min)
[Ca²¹], (a.u.)
30 40
40
2.5
distance between Na,K-ATPase and InsP3R could be somewhat
larger than the working distance for FRET fluorophores, GFP
and Cy3. Hence, the FRET results do not rule out the possibil-
ity that Na,K-ATPase and InsPÅR may interact via a scaffold-
ing protein. The observed increase in FRET between Na,K-
ATPase and InsP3R following ouabain exposure and the loss of
FRET upon CytD treatment indicate that ouabain-induced
Ca²+ oscillations were dependent on a dynamic physical asso-
ciation between Na,K-ATPase and InsP3R and therefore add
further evidence to the concept that the Ca²+ signal arises from
a signaling microdomain containing Na,K-ATPase and InsPR.
The question remains as to how the localization of Na,K-
ATPase and InsP3R is controlled. As evidenced by our FRET
and co-immunoprecipitation studies using CytD, an intact cy-
toskeleton is required for the physical association between
Na,K-ATPase and InsP3R and ouabain-induced Ca²+ signal-
ing. This suggests that actin, or cytoskeletal proteins associ-
ated with actin, will stabilize the Na,K-ATPase/InsP3R com-
plex by physical tether cross-linking. Ankyrins, a family of
adaptor proteins believed to participate in the organization of
proteins into specialized regions in the plasma membrane and
ER Ca²+ stores, represent an interesting putative mechanistic
partner for orchestrating Na,K-ATPase and InsP3R proximity
(25). The complete framework of adaptor and scaffolding pro-
teins that may be involved in facilitating the structure and
signal-transducing function of this microdomain is still uncer-
MEVSMDDHKLSLDELHRKYGTDLSRGLTPARAAEILARDGPNALTPPPTTPEWVKFCRQ...
2.0
1.5
1.0
50
20
Time (min)
FIG. 5. Role of NH₂ terminus of Na,K-ATPase al-subunit in ouabain-induced Ca²+ signaling. a, NH, terminus of the rat Na,K-ATPase
al-subunit and deletion mutant thereof. Numbering is based on the mature rat al-subunit amino acid sequence (47). b, confocal microscope image
of a GFP-NKAa1.M32-expressing RPT cell, recorded with a small pinhole to optimize membrane signal. c, intracellular Na+ measurements
following ouabain treatment (at time 0) in RPT cells expressing GFP-NKAa1.M32 (trace A) and endogenous Na,K-ATPase al-subunit (trace B).
Arbitrary units (a.u.) represent ratio values corresponding to intracellular Ca²+ and Na+ concentration changes. d, single cell recordings of
intracellular Ca²+ response to ouabain in GFP-NKAa1.M32-expressing RPT cells. No Ca²+ oscillations were observed. e, GST pull-down assay with
RPT cell lysate followed by Western blotting for InsP3R3.
0
60
10
NF-kB
activation
control)
NF-kB
(fold
70
30
80
1.4
1.3
1.2
1.1
1.0
0.9
90
2
1: GST-NKAa1.N95
2: GST
*
0.8
GFP-NKAα1.M32
NKA 1.M32
+
FIG. 6. NF-kB translocation in GFP-NKAa1.M32-expressing cells following ouabain treatment. a, GFP-NKAa1.M32-expressing RPT
cell identified by GFP fluorescence. Images were recorded with an open pinhole to measure semiquantitatively the ratio between NF-kB abundance
in the nucleus and cytosol. b, NF-kB immunosignal of GFP-NKAa1.M32-expressing and non-expressing RPT cells, from the same field of view as
a. c, NF-kB activation in GFP-NKAa1.M32-expressing (n = 47) and non-expressing (n = 218) cells following ouabain treatment, mean ± S.E., *,
p < 0.05. Ouabain-induced NF-kB nuclear translocation was abrogated in GFP-NKAa1.M32-expressing cells.
tain, but our results also do not preclude the necessity of a
direct physical interaction between Na,K-ATPase and InsPR
in this event. The activation of InsP³R is likely due to an
allosteric effect of ouabain on Na,K-ATPase. Na,K-ATPase is a
P-type ATPase that can exist in distinct E₁ and E₂ conforma-
tional states that are at least partially determined by intramo-
lecular interactions between the NH₂ terminus and cytoplas-
mic loops of the enzyme. Ouabain binds to the E₂ conformation
of the Na,K-ATPase and causes a robust shift in the E₁/E2 poise
toward E₂ forms (36). Information derived from the crystal
structure of another P-type ATPase, the SERCA pump, sug-
gests that the E₁ E₂ state transition is accompanied by
significant movement of the three cytoplasmic domains, N (nu-
cleotide binding), P (phosphorylation), and A (actuator) (37). A
recent study (28) suggests that the NH₂ terminus may act as an
auto-regulatory domain, modulating E₁/E₂ conformational
transition. In E₁ conformation, the NH₂ terminus is in associ-
ation with the first cytoplasmic loop of Na,K-ATPase. Transi-
tion from E₁ to E2 conformation may release the NH₂ terminus
from its interaction with the first cytoplasmic loop of Na,K-
ATPase, thus making the NH₂ terminus available for interac-
tion either with the InsP3R directly or with a protein bridging
between Na,K-ATPase and InsP3R. Such an effect could ex-
plain why truncation of the first 32 amino acids of the NH₂
terminus will prevent ouabain-induced Ca²+ oscillations. An
alternative explanation is that NH₂-terminal truncation has](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F10b2558f-14bb-4a79-9eba-8554e0210a1c%2F32af8b42-0a77-475f-bd40-dc10e67a553e%2Fuvjt9jv_processed.jpeg&w=3840&q=75)
Transcribed Image Text:50360
NKAa1 (MGKGV)
NKAa1.M32
b
GFP-NKAa1.M32
Na,K-ATPase and InsP3 Receptor in a Signaling Microdomain
30
GRDKYEPAAVSEHGDKKSKKAKKERDMDELKKEVSMDDHKLSLDELHRKYGTDLSRGLT PARAAEILARDGPNALTPPPTTPEWVKFCRQ...
a
1
[Na], (a.u.)
1.6
1.4
1.2
1.0
10
-10
20
0
10 20
Time (min)
[Ca²¹], (a.u.)
30 40
40
2.5
distance between Na,K-ATPase and InsP3R could be somewhat
larger than the working distance for FRET fluorophores, GFP
and Cy3. Hence, the FRET results do not rule out the possibil-
ity that Na,K-ATPase and InsPÅR may interact via a scaffold-
ing protein. The observed increase in FRET between Na,K-
ATPase and InsP3R following ouabain exposure and the loss of
FRET upon CytD treatment indicate that ouabain-induced
Ca²+ oscillations were dependent on a dynamic physical asso-
ciation between Na,K-ATPase and InsP3R and therefore add
further evidence to the concept that the Ca²+ signal arises from
a signaling microdomain containing Na,K-ATPase and InsPR.
The question remains as to how the localization of Na,K-
ATPase and InsP3R is controlled. As evidenced by our FRET
and co-immunoprecipitation studies using CytD, an intact cy-
toskeleton is required for the physical association between
Na,K-ATPase and InsP3R and ouabain-induced Ca²+ signal-
ing. This suggests that actin, or cytoskeletal proteins associ-
ated with actin, will stabilize the Na,K-ATPase/InsP3R com-
plex by physical tether cross-linking. Ankyrins, a family of
adaptor proteins believed to participate in the organization of
proteins into specialized regions in the plasma membrane and
ER Ca²+ stores, represent an interesting putative mechanistic
partner for orchestrating Na,K-ATPase and InsP3R proximity
(25). The complete framework of adaptor and scaffolding pro-
teins that may be involved in facilitating the structure and
signal-transducing function of this microdomain is still uncer-
MEVSMDDHKLSLDELHRKYGTDLSRGLTPARAAEILARDGPNALTPPPTTPEWVKFCRQ...
2.0
1.5
1.0
50
20
Time (min)
FIG. 5. Role of NH₂ terminus of Na,K-ATPase al-subunit in ouabain-induced Ca²+ signaling. a, NH, terminus of the rat Na,K-ATPase
al-subunit and deletion mutant thereof. Numbering is based on the mature rat al-subunit amino acid sequence (47). b, confocal microscope image
of a GFP-NKAa1.M32-expressing RPT cell, recorded with a small pinhole to optimize membrane signal. c, intracellular Na+ measurements
following ouabain treatment (at time 0) in RPT cells expressing GFP-NKAa1.M32 (trace A) and endogenous Na,K-ATPase al-subunit (trace B).
Arbitrary units (a.u.) represent ratio values corresponding to intracellular Ca²+ and Na+ concentration changes. d, single cell recordings of
intracellular Ca²+ response to ouabain in GFP-NKAa1.M32-expressing RPT cells. No Ca²+ oscillations were observed. e, GST pull-down assay with
RPT cell lysate followed by Western blotting for InsP3R3.
0
60
10
NF-kB
activation
control)
NF-kB
(fold
70
30
80
1.4
1.3
1.2
1.1
1.0
0.9
90
2
1: GST-NKAa1.N95
2: GST
*
0.8
GFP-NKAα1.M32
NKA 1.M32
+
FIG. 6. NF-kB translocation in GFP-NKAa1.M32-expressing cells following ouabain treatment. a, GFP-NKAa1.M32-expressing RPT
cell identified by GFP fluorescence. Images were recorded with an open pinhole to measure semiquantitatively the ratio between NF-kB abundance
in the nucleus and cytosol. b, NF-kB immunosignal of GFP-NKAa1.M32-expressing and non-expressing RPT cells, from the same field of view as
a. c, NF-kB activation in GFP-NKAa1.M32-expressing (n = 47) and non-expressing (n = 218) cells following ouabain treatment, mean ± S.E., *,
p < 0.05. Ouabain-induced NF-kB nuclear translocation was abrogated in GFP-NKAa1.M32-expressing cells.
tain, but our results also do not preclude the necessity of a
direct physical interaction between Na,K-ATPase and InsPR
in this event. The activation of InsP³R is likely due to an
allosteric effect of ouabain on Na,K-ATPase. Na,K-ATPase is a
P-type ATPase that can exist in distinct E₁ and E₂ conforma-
tional states that are at least partially determined by intramo-
lecular interactions between the NH₂ terminus and cytoplas-
mic loops of the enzyme. Ouabain binds to the E₂ conformation
of the Na,K-ATPase and causes a robust shift in the E₁/E2 poise
toward E₂ forms (36). Information derived from the crystal
structure of another P-type ATPase, the SERCA pump, sug-
gests that the E₁ E₂ state transition is accompanied by
significant movement of the three cytoplasmic domains, N (nu-
cleotide binding), P (phosphorylation), and A (actuator) (37). A
recent study (28) suggests that the NH₂ terminus may act as an
auto-regulatory domain, modulating E₁/E₂ conformational
transition. In E₁ conformation, the NH₂ terminus is in associ-
ation with the first cytoplasmic loop of Na,K-ATPase. Transi-
tion from E₁ to E2 conformation may release the NH₂ terminus
from its interaction with the first cytoplasmic loop of Na,K-
ATPase, thus making the NH₂ terminus available for interac-
tion either with the InsP3R directly or with a protein bridging
between Na,K-ATPase and InsP3R. Such an effect could ex-
plain why truncation of the first 32 amino acids of the NH₂
terminus will prevent ouabain-induced Ca²+ oscillations. An
alternative explanation is that NH₂-terminal truncation has
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