Calcium Signals Within Membrane Nanotubes

描述（由申请人提供）：细胞间通信是多细胞生物协调功能的绝对要求，细胞早就知道使用间隙连接和突触与它们的邻居进行通信。最近发现了一种通过隧道膜纳米管（TNTs）进行细胞间通信的新途径。这些是动态的膜突起，直径几百纳米，在几十微米的距离上物理地连接细胞体，并允许细胞质分子、膜成分甚至细胞器在邻近细胞之间交换。Ca2+信号沿tnt的传递被认为是细胞间通信的一种手段，它可以调节多种生理过程，如基因表达、酶活性和电兴奋性。我们的模型研究表明，Ca2+离子沿tnt的被动扩散不足以支持细胞间Ca2+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tnt长度内产生的局部自发和肌醇三磷酸诱发的Ca2+信号的观察表明，细胞间Ca2+信号沿着tnt积极传播的机制。因此，我们假设Ca2+激活的Ca2+释放通道簇作为扩增位点，以克服动作电位沿轴突电传输的化学模拟中的被动扩散限制。我们的总体目标是阐明这种Ca2+波沿tnt传播的新机制的机制，并探索其在Ca2+信号的生理和病理细胞间通信中的作用。我们的具体目标是；(1)利用荧光钙指示剂，我们将确定tnt内再生局部Ca2+释放事件中涉及的Ca2+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Ca2+释放通道，并利用单分子超分辨率成像，我们将探索IP3Rs的扩散运动和内质网沿其长度的连续性质。(3)我们将探讨局部钙释放事件如何相互协调以传播Ca2+波，这一过程有效发生的要求，并研究tnt在响应细胞应激时传播异常Ca2+信号的作用。我们的建议将为tnt介导的Ca2+信号传播提供重要的机制见解，并可能导致我们对这种细胞间通讯新机制所涉及的生理和病理生理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.