Calcium Signals Within Membrane Nanotubes

描述(申请人提供)：细胞间通讯是多细胞生物体协调运作的绝对要求，细胞很早就知道利用缝隙连接和突触与邻居通讯。最近，通过隧道膜纳米管(TNTs)发现了一种新的细胞间通讯途径。这些是直径几百纳米的动态膜突起，它们在物理上将细胞体连接在数十微米的距离上，并允许胞浆分子、膜组件甚至相邻细胞之间的细胞器交换。钙信号沿TNTs的传递被认为是细胞间通讯的一种方式，它可以调节基因表达、酶活性和电兴奋性等多种生理过程。我们的模拟研究表明，钙离子在TNTs上的被动扩散不足以支持细胞间钙信号的有效传输。相反，我们对在培养的哺乳动物细胞之间形成的TNTs长度内产生的局部自发和三磷酸肌醇诱发的钙信号的观察表明，细胞间钙信号沿着TNTs活跃传播的机制。因此，我们假设，钙激活的钙释放通道簇起到放大作用，以克服被动扩散的限制，在化学模拟动作电位沿轴突的电传递中。我们的总体目标是阐明这种新的钙波沿TNTs传播的机制，并探索其在生理和病理细胞间钙信号交流中的作用。我们的具体目标是：(1)使用荧光钙指示剂，我们将确定参与TNTs内再生局部钙释放事件的钙通道的机制和类型，并阐明在时间和空间上塑造这些局部事件的隔离/缓冲系统。(2)利用新的超分辨率成像技术，我们将沿着TNT以纳米精度绘制单个钙释放通道，并利用单分子超分辨率成像，探索IP3R的扩散运动和内质网沿其长度的毗连性质。(3)我们将探索局部钙释放事件如何相互协调以传播钙波，以及这一过程有效发生的要求，并研究TNTs在传播异常的钙信号以响应细胞应激中的作用。我们的建议将为TNT介导的钙信号的传播提供重要的机制见解，并可能导致我们对这一新的细胞间通讯机制所涉及的生理和病理生理过程的理解取得重大进展。 与公共健康相关：细胞之间相互交流信息的能力对正常生理至关重要，众所周知，细胞之间处理信息的错误会导致癌症、阿尔茨海默氏症和糖尿病等疾病。在这里，我们建议研究一种新的细胞间通讯形式，其中钙被用来在细胞膜的很长、很细的管状延伸内直接传递信息。我们的建议旨在为这一过程是如何发生的提供重要的机械论见解，并可能导致我们对细胞间通信所涉及的生理学和病理生理学过程的理解取得重大进展。

DESCRIPTION (provided by applicant): Intercellular communication is an absolute requirement for the coordinated functioning of multi-cellular organisms, and cells have long been known to employ gap junctions and synapses to communicate with their neighbors. A new route of cell-to-cell communication has recently been identified via tunneling membrane nanotubes (TNTs). These are dynamic membrane protrusions, a few hundred nanometers in diameter, that physically link cell bodies over distances of tens of microns and allow for the exchange of cytosolic molecules, membrane components and even organelles between neighboring cells. Transmission of Ca2+ signals along TNTs has been proposed as a means of intercellular communication, which may regulate physiological processes as diverse as gene expression, enzyme activity and electrical excitability. Our modeling studies indicate that passive diffusion of Ca2+ ions along TNTs is inadequate to support efficient transmission of Ca2+ signals between cells. Instead, our observations of local spontaneous and inositol trisphosphate-evoked Ca2+ signals generated within the length of TNTs formed between cultured mammalian cells suggest a mechanism for active propagation of intercellular Ca2+ signals along TNTs. We thus hypothesize that clusters of Ca2+- activated Ca2+ release channels function as amplification sites to overcome limitations of passive diffusion in a chemical analog of electrical transmission of action potentials along axons. Our overall goals are to elucidate the mechanisms underlying this novel mechanism of Ca2+ wave propagation along TNTs, and to explore its role in the physiological and pathological cell-cell communication of Ca2+ signals. Our specific aims are; (1) Using fluorescent calcium indicators we will determine the mechanisms and types of Ca2+ channels involved in regenerative local Ca2+ release events within TNTs, and elucidate the sequestration/buffering systems that shape these localized events in time and space. (2) Utilizing novel superresolution imaging techniques we will map individual Ca2+ release channels with nanometer precision along the TNT and, employing single molecule superresolution imaging, we will explore the diffusional motility of IP3Rs and the contiguous nature of the endoplasmic reticulum along its length. (3) We will explore how local calcium release events coordinate with one another to propagate a Ca2+ wave, the requirements for this process to occur efficiently, and investigate the role for TNTs in spreading aberrant Ca2+ signals in response to cellular stress. Our proposal will provide important mechanistic insights into TNT-mediated propagation of Ca2+ signals and will likely lead to significant advances in our understanding of the physiology and pathophysiological processes involved in this novel mechanism of cell-to-cell communication. PUBLIC HEALTH RELEVANCE: The ability of cells to communicate information between one another is crucial for normal physiology and errors in processing information between cells are known to contribute to diseases such as cancer, Alzheimer's disease and diabetes. Here we propose to study a new form of communication between cells where calcium is used to transmit information directly within very long, thin, tubular extensions of the cell membrane. Our proposal aims to provide important mechanistic insights into how this process occurs and will likely lead to significant advances in our understanding of the physiology and pathophysiological processes involved in cell- to-cell communication.