DeFazio Richard A, Dvoryanchikov Gennady, Maruyama Yutaka, Kim Joung Woul, Pereira Elizabeth, Roper Stephen D, Chaudhari Nirupa
Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
J Neurosci. 2006 Apr 12;26(15):3971-80. doi: 10.1523/JNEUROSCI.0515-06.2006.
Taste buds are aggregates of 50-100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C beta2 (PLCbeta2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCbeta2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCbeta2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (alpha1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCbeta2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells.
味蕾是由50 - 100个细胞组成的聚集体，其中只有一小部分表达味觉受体和细胞内信号蛋白的基因。我们将功能性钙成像与单细胞分子谱分析相结合，以证明小鼠味蕾中存在两种不同的细胞类型。钙成像显示，分离的味觉细胞对味觉刺激物或用氯化钾进行去极化处理时，细胞质Ca2+会短暂升高，但不会同时对两者都有反应。通过单细胞逆转录（RT）-PCR，我们发现单个味觉细胞要么表达磷脂酶Cβ2（PLCβ2）（一种必需的味觉转导效应器），要么表达突触体相关蛋白25（SNAP25）（钙触发递质胞吐作用的关键成分）。钙成像揭示的这两种功能类别与单细胞RT-PCR确定的两种基因表达类别相对应。具体而言，对味觉刺激物有反应的细胞表达PLCβ2，而对氯化钾去极化有反应的细胞表达SNAP25。我们通过两种方法证明了这一点：第一，通过顺序钙成像和单细胞RT-PCR；第二，对来自转基因小鼠切片中的味蕾进行钙成像，在这些转基因小鼠中，表达PLCβ2的味觉细胞用绿色荧光蛋白标记。为了评估表达SNAP25的细胞的重要性，我们从单细胞进行RNA扩增，然后进行RT-PCR。我们发现SNAP25阳性细胞还表达典型的突触前蛋白，包括电压门控钙通道（α1A）、神经细胞黏附分子、突触素-II以及神经递质合成酶谷氨酸脱羧酶和芳香族氨基酸脱羧酶。通过扩增RNA谱分析或免疫细胞化学在PLCβ2细胞中均未检测到突触标记物。这些数据证明了至少存在两种分子上不同的功能性味觉细胞类别：受体细胞和形成突触的细胞。
