Is the novel rhythmically expressed gene 'quasimodo' the missing link between the circadian clock and membrane properties of pacemaker neurons?

生物钟驱动着包括人类在内的许多生物体的生物节律。它们调节我们的睡眠/觉醒周期，体温以及生理和行为的许多其他方面。我们的主生物钟位于大脑中，在一个称为视交叉上核（SCN）的结构中。它由成千上万的神经元组成，它们都表达一组所谓的“时钟基因”。这些基因是特殊的，因为它们通过反馈回路相互调节，这意味着一个基因的产物抑制另一个基因甚至其自身的表达。因此，可以观察到这些基因中的许多基因的分子振荡，其发生频率约为1000。24小时：我们的昼夜分子钟工作！但这还不足以实现昼夜节律的行为：例如，为了确定我们是否应该活动或睡觉，SCN神经元必须以某种方式向我们大脑和身体的其他区域发出一天中的时间信号，例如松果体，当SCN这样说时，松果体会产生“睡眠激素”褪黑激素。SCN（或起搏器）神经元通过以有节奏的方式产生电信号（动作电位）来做到这一点;某种程度上受到分子钟的调节。但这是怎么做到的呢？最近的证据表明，神经元膜的性质是由分子钟有节奏地改变，但同样，问题仍然是如何实现的。在我们的模式生物果蝇中，情况非常相似。它的大脑中也有起搏神经元，以昼夜节律的方式表达许多时钟基因。我们最近分离出一种新的节律性表达基因quasimodo（qsm），它存在于一些生物钟神经元中，可能附着在它们的细胞膜外。当我们通过实验改变qsm的表达水平时（与正常果蝇相比或多或少），我们诱导的效果与操纵基因改变神经元电活动（称为离子通道）的效果非常相似。因此我们相信qsm至少与这些基因中的一部分相互作用，并且因为它的表达是由分子钟控制的，qsm很可能是连接分子钟和神经元膜的一个因子！为了找出这一点，我们想确定哪些离子通道蛋白与Qsm蛋白相互作用，以及当我们影响它们的表达时到底发生了什么。我们还注意到，在操纵神经元膜蛋白的某些情况下，我们可以在没有时钟基因的情况下观察到强烈的行为节律，而时钟基因一直被认为是这种节律的绝对必要条件。这是非常令人惊讶的，这意味着神经元膜具有内在的节律产生特性。我们将尝试找出是什么产生了这些节奏，通过执行诱变筛选，我们将寻找突变体，取消异常节奏的苍蝇的节奏。我们的想法是，有两种节律产生机制：已知的一种由时钟基因及其产物组成，另一种在神经元膜中运作。我们认为它们是相互联系的，通过本提案中概述的实验，我们希望展示时钟基因如何控制膜振荡器，以及膜振荡器如何反过来反馈控制时钟基因。

Circadian clocks drive biological rhythms in many organisms including humans. They regulate our sleep/wake cycle, body temperature, and many other aspects of physiology and behaviour. Our master circadian clock resides in the brain, in a structure called the Supra Chiasmatic Nuclei (SCN). It consists of thousands of neurons which all express a set of so called 'clock genes'. These genes are special, since they regulate each other via feedback loops, meaning that the product of one gene represses another gene or even its own expression. As a result, molecular oscillations of many of these genes can be observed that occur with a frequency of ca. 24 hrs: our circadian molecular clock work! But this is not enough to achieve a circadianly rhythmic behaviour: For example to determine if we should be active or go to sleep, the SCN neurons somehow have to signal the time-of-day to other regions of our brain and body, for example to the pineal gland, which produces the 'sleeping hormon' melatonin when the SCN says so. SCN (or pacemaker) neurons do this, by producing electric signals (action potentials) in a rhythmic fashion; somehow regulated by the molecular clock. But how does this work? Recent evidence indicates that the properties of the neuronal membrane are rhythmically changed by the molecular clock, but again, the question remains how this is accomplished. In our model organism, the fruit fly Drosophila melanogaster, the situation is quite similar. It also has pacemaker neurons in its brain which express many clock genes in a circadian fashion. We recently isolated a novel rhythmically expressed gene called quasimodo (qsm), which is present in some of the clock neurons and is probably attached to the outside of their membranes. When we experimentally change the expression level of qsm (make more or less of it compared to normal flies), we induce very similar effects as if we manipulate genes that alter the electrical activity of neurons (called ion-channels). Therefore we believe that qsm interacts with at least some of these genes, and because its expression is controlled by the molecular clock, qsm could well be a factor that connects the clock with the neuronal membrane! To find this out we want to identify, which of the ion channel proteins interact with the Qsm protein and what exactly happens when we influence their expression. We also noticed that under certain circumstances of manipulating neuronal membrane proteins, we can observe strong behavioural rhythms in the absence of clock genes, which always were thought to be absolutely required for such rhythms. This is very astonishing and implies that there are rhythm-generating properties intrinsic to the neuronal membrane. We will try to find out what generates these rhythms by performing a mutagenesis screen where we will look for mutants that abolish the rhythms in the abnormally rhythmic flies. Our idea is that there are two rhythm-generating mechanisms: The known one consisting of the clock genes and their products, and another one, operating in the neuronal membranes. We think they are connected to each other, and with the experiments outlined in this proposal we want to show how the clock genes control the membrane oscillator and how in turn the membrane oscillator feeds back to control the clock genes.