Transcript Dr. Kathryn Medler

CELL SIGNALING PROCESSES
IN TASTE CELLS
KATHRYN MEDLER LAB
Why should we care about taste?
Taste is used to determine if potential food items will be ingested
or rejected.
Taste is used by all organisms and is the oldest sensory system.
Loss of taste can lead to depression and loss of appetite which
can cause malnutrition. Deficits in the taste system can also lead
to uncontrolled appetite and obesity.
Mammalian taste buds are present in
papillae on the tongue
Taste stimuli
Apical
Taste bud
Basolateral
Afferent gustatory
nerve fiber
Lingual
Epithelium
Taste Transduction: Two distinct
signaling pathways exist
Bitter
Sweet
Umami
+
Na+ H
Multiple signaling
pathways are present
taste cells and all
pathways depend on
increases in
intracellular calcium to
transmit signals to the
nervous system.
R
PLC
+
++
IP3
Ca2+
Store
Na+
Ca2+
K+
[Ca2+]
Serotonin
TRPM5
[Ca2+]i
i
ATP
Our lab is studying these
different signaling
mechanisms: how they
function and how they
are regulated.
Calcium imaging measures changes in calcium
levels in live cells
We can characterize the functional role of different proteins
expressed in these cells and how they affect calcium signals.
Using calcium imaging we found:
“Ionic” stimuli
“Complex” stimuli
1750
hi K
Bitter
1500
hi K
1500
1250
1250
[Ca2+]i nM
[Ca2+]i nM
Bitter
1750
1000
750
1000
750
500
500
250
250
0
0
0
100
200
300
Time (s)
Activate calcium release from stores
0
100
200
300
400
Time (s)
Activate voltage gated calcium influx
Different taste stimuli evoke different signals.
Hacker et al., 2008
Surprisingly, we also found
1250 Bitter
hi K
Some taste cells
responded to bitter
stimuli AND cell
depolarization.
750
2+
[Ca ]i (nM)
1000
500
250
0
0
200
400
Time (s)
600
Hacker et al., 2008
This is a newly identified sub-population of taste cells.
We asked the question:
How do these taste cells respond
to multiple stimuli?
Expression patterns of PLCb3/IP3R1 in taste cells
D
PLCb3 and IP3R1 are co-expressed in a population of taste cells that are distinct
from the PLCb2 expressing cells.
Further studies are being conducted to characterize
this newly identified signaling pathway.
There are 3 separate taste cell
groups.
+
Na
+
H
++
+ ++
+ +
+ ++
+ +
+ +
Taste stimuli
Taste stimuli
b
 gustducin
G proteins
Phospholipase Cb3
Phospholipase Cb2
IP3
IP3
IP3R3
IP3R1
Ca2+
Endoplasmic
reticulum
Endoplasmic
reticulum
Na+
Ca2+
K+
[Ca2+]i
Ca2+
+ +
+
+
++ +
+
++
+
+ + +
+
ATP
TRPM5
[Ca2+]i
?
Na+
Ca2+
hemichannel
We are asking “How do each of these groups
contribute to detection of taste stimuli?”
We are also studying the evoked taste
responses in obese mice.
We asked “Are peripheral taste responses different in
obese mice versus normal mice?”
Norm
Obese
NS
50
NS
25
***
***
***
100
Percent Increase over Baseline
Percentage of Responsive Cells
75
Norm
Obese
75
50
25
***
**
***
0
0
MPG
Sac
Ace K
Den
hi K
MPG
Sac
AceK
Den
The number of responsive taste cells and the response
amplitudes are reduced in obese mice for the appetitive
tastes.
We’re asking how does this affect the animal’s ability to
perceive taste stimuli? Is it reversible?
We recently identified a new
TRP channel in taste cells.
A
100
DEN
TPPO
+DEN
B
1000
Arbitrary Units
TRPM5 is a well-known monovalent selective TRP
channel that is important in taste transduction. TRPM5
turns on in response to some taste stimulation.
500
9PHE+
DEN
DEN
DEN
DEN
Arbitrary Units
75
50
25
0
0
0
200
400
Time (s)
600
0
200
400
600
Time (s)
We found that taste cells also express TRPM4, which is
the other monovalent selective TRP channel. TRPM4 is
also activated by some taste stimuli.
We are determining the role of
TRPM4 in taste transduction.
2+
110
Ca
+
Na
DEN
20
10
90
0
0
100
200
Time (s)
300
400
Arbitrary Units
100
2+
[Ca ]i (nM)
Hi K
In some cells, taste
stimuli evoke sodium
and calcium
increases. Using
imaging and patch
clamp, we’re
determining how
TRPM4 contributes
to these responses.
Gene regulation by WT1
in taste cells
In collaboration with Stefan Roberts lab
Transcriptional Regulation by WT1
BASP1
General transcription
machinery
IIH
IIF
WT1
IIA
IIB
IID
IIE
Pol II
TATA
Growth factors
Amphiregulin
IGFII
PDGF-A
Apoptosis
Bcl 2
Bak
c-myc
Differentiation
Podocalyxin
Nephrin
WT1 plays a critical role in the development
of several organs and tissues
WT1 Knock-out mice
Kidneys
Gonads
Spleen
Adrenal glands
Diaphragm
Retinal Ganglia
Olfactory epithelium
Taste buds
WT1 and BASP1 are expressed in taste cells
Embryonic
Adult
WT1
Ctrl
BASP1
Ctrl
E13
E14.5
WT1
E17.5
WT1 null mice fail to develop a peripheral taste system
WT
WT
Troma 1
E13.5
Sox2
KO
KO
WT
WT
GAP-43
Shh
KO
KO
WT1 regulates genes critical for taste cell development
Real time PCR
1.2
LEF1
0.8
0.6
0.4
0.2
6
0
LEF1
5
WT
KO
4
3
2
*
1.2
0
IgG
WT1
BASP1
Relative mRNA
1
PTCH1
1
0.8
0.6
0.4
0.2
0
7
6
WT
PTCH1
KO
5
4
1.2
3
2
1
0
IgG
WT1
BASP1
Relative mRNA
Fold Enrichment
Fold Enrichment
*
1
Relative mRNA
CHiP assay
*
1
0.8
0.6
0.4
0.2
0
WT1+/+
WT1-/-
BMP4
Primary taste cells can be cultured and transfected
CHiP
qPCR
14
Hoechst
Hoechst
Hoechst
WT1
PLCβ2
1.2
LEF1
WT1
1
10
Relative mRNA
Fold Enrichmet
12
8
6
4
2
TRMP5-GFP
0.8
0.6
0.4
0.2
0
0
IgG
WT1
BASP1
Control
siRNA
3.5
PTCH1
1.2
LEF1
Relative mRNA
2.5
2
1.5
1
0.5
Knockdown of WT1 in
cultured taste buds
causes a reduction in the
expression of WT1 target
genes that are important
in taste cell maintenance.
1
0.8
0.6
0.4
0.2
0
IgG
WT1
BASP1
0
Control
siRNA
WT1
siRNA
1.2
PTCH1
1
Relative mRNA
Fold Enrichment
3
WT1
siRNA
0.8
0.6
0.4
0.2
0
Control
siRNA
WT1
siRNA
Combine the physiological and
molecular approaches of the Medler
and Roberts labs to study the role of
WT1 and BASP1 in gene regulation
during development and tissue
homeostasis
If you are interested in rotating in
the lab on any of these projects,
please contact me by email:
[email protected]