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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]