Genetically Encoded Calcium Indicators in Toxoplasma gondii

描述（由申请人提供）：细胞溶质游离钙（Ca 2+）浓度的波动调节所有真核生物中的多种细胞功能。然而，如果Ca 2+升高超过生理水平，则会对细胞造成损害。细胞包含一套复杂的机制来平衡胞质Ca 2+水平，并且升高胞质中Ca 2+的信号由降低其的机制来补偿。这些Ca 2+依赖性稳态机制的改变是人类许多突出疾病的原因，例如心力衰竭或神经元死亡。顶复门寄生虫包括许多医学和兽医相关的病原体。Ca 2+调节和稳态在弓形虫感染周期中的作用已得到很好的证明。也有一些关于Ca 2+在疟原虫属生命周期中的作用的信息。在弓形虫中，Ca 2+信号参与刺激微线分泌、滑行运动、锥体挤出和侵入。然而，关于这是如何机械地发生的以及所涉及的分子的信息是碎片化的或缺失的。此外，绝大多数已知的Ca 2+相关的基因仍然是未知的。一个有趣的情况是，实验证据的存在肌醇1，4，5-三磷酸（IP 3）依赖的钙反应在疟原虫属。和T.弓形虫，但没有IP 3（IP 3R）或兰尼碱（RyR）受体直系同源物已被确定在这些基因组中的任何一个。我们自己的初步数据支持存在一个高度调节的机制Ca 2+进入，但这些调控分子的候选基因是未知的。在T.弓形虫，而不是疟原虫属。或隐孢子虫属（Cryptosporidium spp.）基因组关于顶复门中Ca 2+储存和功能的可用信息，虽然是碎片化的，但指向这些寄生虫中存在独特的Ca 2+介导的途径。Ca ~（2+）在T.使用间接方法如用荧光染料标记细胞外寄生虫或使用细胞内Ca 2+螯合剂和/或离子载体获得弓形虫。我们的工具将允许执行直接实时观察的Ca 2+的变化在T。弓形虫滑行运动、锥体挤出、微丝分泌和宿主细胞侵入和排出。我们建议生成T.表达遗传编码Ca 2+指示剂（GECI）的弓形虫速殖子靶向不同的细胞区室，以揭示活寄生虫中Ca 2+的动态，并探索宿主细胞入侵和外出对Ca 2+信号的要求。弓形虫是理想的系统，因为它的遗传易处理性，分离克隆系的容易性和其明确的响应Ca 2+使这些工具的验证是可行的。这些细胞将允许进行直接测量，并将在细胞内速殖子中进行可行的研究。我们将能够研究的作用，Ca 2+在宿主细胞的出口和生理Ca 2+通量之间的不同Ca 2+商店。

DESCRIPTION (provided by applicant): Fluctuations of the cytosolic free calcium (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. However, if Ca2+ elevates over the physiological level it becomes damaging to the cell. Cells contain a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in these Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, as for example heart failure or neuronal death. Apicomplexan parasites include a number of pathogens of medical and veterinary relevance. The role of Ca2+ regulation and homeostasis in the infection cycle of Toxoplasma gondii has been very well documented. There is also some information on the role of Ca2+ in the life cycle of Plasmodium spp. In Toxoplasma, Ca2+ signaling is involved in the stimulation of microneme secretion, gliding motility, conoid extrusion and invasion. However, the information on how this happens mechanistically and the molecules involved is fragmented or missing. In addition, the vast majority of the known Ca2+-related genes remain uncharacterized. An interesting case is that the presence of experimental evidence for an inositol 1,4,5- trisphosphate (IP3)-dependent Ca2+ response in Plasmodium spp. and T. gondii, but no IP3 (IP3R) or ryanodine (RyR) receptor orthologs have been identified in either of those genomes. Our own preliminary data supports the presence of a highly regulated mechanism for Ca2+ entry but the candidate genes for these regulatory molecules are not known. Several potential Ca2+ channels are present in T. gondii but not in Plasmodium spp. or Cryptosporidium spp. genomes. The available information on Ca2+ storage and function in Apicomplexans, although fragmented, point toward the presence of unique Ca2+-mediated pathways in these parasites. The information on the role of Ca2+ in the lytic cycle of T. gondii has been obtained using indirect methods such as labeling of extracellular parasites with fluorescent dyes or using intracellular Ca2+ chelators and/or ionophores. Our tools will allow performing direct real-time observations of Ca2+ changes during T. gondii gliding motility, conoid extrusion, microneme secretion and host-cell invasion and egress. We propose to generate T. gondii tachyzoites expressing Genetically Encoded Ca2+ Indicators (GECI) targeted to different cellular compartments to reveal the dynamics of Ca2+ in live parasites and explore the requirements for Ca2+ signaling in host-cell invasion and egress. Toxoplasma is the ideal system because of its genetic tractability, the easiness of isolating clonal lines and its clear-cut response to Ca2+ making validation of these tools feasible. These cells will allow performing direct measurements and will make feasible studies in intracellular tachyzoites. We will be able to study the role of Ca2+ in host-cell egress and physiological Ca2+ fluxes between different Ca2+ stores.