Phosphoinositide cycle in Drosophila

果蝇的磷酸肌醇循环

• 批准号: BB/M007006/1
• 负责人: Roger Hardie
• 金额: $ 78.62万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM007006%2F1
• 关键词: Phosphoinositide; cycle; Drosophila

Photoreceptors transduce light into electrical signals by a series of biochemical steps, each involving specific protein molecules (e.g. visual pigments and enzymes). The end result of this "phototransduction cascade" is the activation of proteins known as "ion channels", in the lipid membrane surrounding the cell. Once activated, ion channels open to allow charged ions, such as sodium and calcium, into the cell, thereby generating electrical signals for transmission to the brain. Phototransduction can be particularly well studied in the fruitfly Drosophila because of the ease with which we can manipulate specific genes (and hence proteins) and because we can record the activity from their photoreceptors with a range of high precision techniques. The molecules involved are not unique to fly photoreceptors and closely related molecules are found in cells throughout our own bodies. One such molecule is the so-called TRP channel. In flies, this is the channel activated during phototransduction; in mammals, TRP channels are essential for a wide range of vital processes such as hormonal responses, regulation of blood pressure, taste, smell, and sensations of pain, hot and cold. The particular cascade used by the fly photoreceptor to activate the TRP channels is called the phospho-inositide (PI) cycle. This is one of the most widely used biochemical cascades in living cells. In humans it is found in almost every cell in the body and is responsible for a wide range and hormonal responses, such as those involved in regulating blood pressure as well as in communication between neurons in the brain and various senses, such as taste. Activation of this cascade involves the breakdown of an important lipid molecule found in all cell membranes known as PIP2. To maintain operation it is essential that PIP2 is continually resynthesised. This takes place via a complex cycle involving multiple steps and at least 5 distinct intermediates. If any of these steps is compromised, not only does the cascade cease working, but the cells affected can die or become cancerous. In order to study this cycle in living cells, researchers have developed fluorescently labelled molecules which bind to each intermediate which can then be viewed with special microscopes. Normally such experiments are performed under artificial conditions in cultured cells which may differ in their behaviour to cells in living tissue. In our research we will use genetic techniques to express a range of such fluorescently labelled sensors in fly photoreceptors. By exploiting some unique optical features of the fly's eye this will enable us to image and measure their fluorescence in the completely intact living animal. This will allow us to follow the fate of the various intermediates in response to physiological stimulation, which in the eye can be precisely controlled by light. Additionally by making mutations in candidate genes, we will also be able to identify the genes responsible for regulating each step of the PI cycle. This approach will allow us to build up a comprehensive and quantitative picture of the functioning of this important and ubiquitous biochemical cycle in the intact animal. The knowledge we gain from these studies will not only further our understanding of how photoreceptors see but, because the basic underlying biochemical mechanisms are so widely found, will provide new insight into many other, often clinically important processes in the body.

光感受器通过一系列生化步骤将光转化为电信号，每个步骤都涉及特定的蛋白质分子(如视觉色素和酶)。这种“光转导级联反应”的最终结果是激活细胞周围脂膜中被称为“离子通道”的蛋白质。一旦被激活，离子通道就会打开，允许带电离子(如钠和钙)进入细胞，从而产生电信号传输到大脑。果蝇的光传导研究特别好，因为我们可以很容易地操纵特定的基因(因此也就是蛋白质)，因为我们可以用一系列高精度的技术记录它们的光感受器的活动。涉及的分子并不是苍蝇光感受器独有的，在我们全身的细胞中都发现了密切相关的分子。其中一个这样的分子就是所谓的Trp通道。在苍蝇中，这是在光传导过程中被激活的通道；在哺乳动物中，色氨酸通道在一系列重要过程中是必不可少的，如激素反应、血压、味道、气味的调节以及疼痛、冷热感觉。苍蝇光感受器用来激活Trp通道的特殊级联被称为磷酸肌醇(PI)循环。这是活细胞中使用最广泛的生化级联之一。在人类中，它几乎存在于身体的每一个细胞中，并负责广泛的范围和激素反应，如参与调节血压的反应，以及大脑中神经元和各种感官之间的沟通，如味觉。这种级联反应的激活涉及到一种重要的脂类分子的分解，这种分子存在于所有细胞膜中，称为PIP2。为了维持运转，必须不断地重新合成PIP2。这是通过一个复杂的循环进行的，包括多个步骤和至少5个不同的中间体。如果这些步骤中的任何一个受到影响，不仅级联反应停止工作，而且受影响的细胞可能会死亡或癌变。为了在活细胞中研究这一循环，研究人员开发了荧光标记的分子，这些分子结合到每个中间体上，然后可以用特殊的显微镜观察。通常，这样的实验是在人工条件下进行的，培养的细胞对活组织中的细胞的行为可能会有所不同。在我们的研究中，我们将使用基因技术在苍蝇光感受器中表达一系列这样的荧光标记传感器。通过利用苍蝇眼睛的一些独特的光学特征，这将使我们能够在完全完整的活动物中成像和测量它们的荧光。这将使我们能够跟踪各种中间体在生理刺激下的命运，而生理刺激在眼睛中可以被光精确地控制。此外，通过对候选基因进行突变，我们还将能够识别负责调节PI周期每一步的基因。这种方法将使我们能够建立一个完整的动物体内这个重要的和普遍存在的生化循环的功能的全面和定量的图景。我们从这些研究中获得的知识不仅将进一步加深我们对光感受器如何观看的理解，而且因为基本的潜在生化机制被广泛发现，将为我们对身体中许多其他通常是临床上重要的过程提供新的见解。

项目成果

Evidence for Dynamic Network Regulation of Drosophila Photoreceptor Function from Mutants Lacking the Neurotransmitter Histamine.

• DOI: 10.3389/fncir.2016.00019
• 发表时间: 2016
• 期刊: Frontiers in neural circuits
• 影响因子: 3.5
• 作者: Dau A;Friederich U;Dongre S;Li X;Bollepalli MK;Hardie RC;Juusola M
• 通讯作者: Juusola M

A Single Residue Mutation in the Gaq Subunit of the G Protein Complex Causes Blindness in Drosophila.

G 蛋白复合物 Gaq 亚基中的单个残基突变导致果蝇失明。

• DOI: 10.17863/cam.23495
• 发表时间: 2018
• 作者: Cao J

Calcium signalling in Drosophila photoreceptors measured with GCaMP6f.

• DOI: 10.1016/j.ceca.2017.02.006
• 发表时间: 2017-07
• 期刊: Cell calcium
• 影响因子: 4
• 作者: Asteriti S;Liu CH;Hardie RC
• 通讯作者: Hardie RC

PCYT1A Regulates Phosphatidylcholine Homeostasis from the Inner Nuclear Membrane in Response to Membrane Stored Curvature Elastic Stress.

• DOI: 10.1016/j.devcel.2018.04.012
• 发表时间: 2018-05-21
• 期刊: Developmental cell
• 影响因子: 11.8
• 作者: Haider A;Wei YC;Lim K;Barbosa AD;Liu CH;Weber U;Mlodzik M;Oras K;Collier S;Hussain MM;Dong L;Patel S;Alvarez-Guaita A;Saudek V;Jenkins BJ;Koulman A;Dymond MK;Hardie RC;Siniossoglou S;Savage DB
• 通讯作者: Savage DB

Calcium signalling in $\textit{Drosophila}$ photoreceptors measured with GCaMP6f

使用 GCaMP6f 测量 $ extit{Drosophila}$ 光感受器中的钙信号传导

• DOI: 10.17863/cam.9292
• 发表时间: 2017
• 作者: Asteriti S