Piezo channels and calcium signaling in Trypanosoma cruzi

克氏锥虫的压电通道和钙信号传导

• 批准号: 10216716
• 负责人: ROBERTO DOCAMPO
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-12 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216716
• 关键词: Area; Biological; Biology; Blood Vessels; Buffers; C-terminal; CRISPR/Cas technology; Calcium Channel; Calcium Signaling; Calcium ion; Cell Compartmentation; Cell membrane; Cells; Cellular Mechanotransduction; Chagas Disease; Characteristics; Chelating Agents; Chemicals; Cytosol; Dependence; Development; Electrophysiology (science); Etiology; Future; Generations; Goals; Growth; Image; Investigation; Knock-out; Lead; Leishmania; Mammalian Cell; Mitochondria; Organism; Orthologous Gene; Parasites; Physiological; Piezo ion channels; Plasmids; Process; Property; Pulmonary Ventilation; Research; Role; Sensory; Signal Transduction; Spottings; Techniques; Time; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Tumor stage; Work; cell motility; environmental change; experimental study; extracellular; genome editing; insight; mechanotransduction; mutant; non-invasive imaging; paralogous gene; prevent; response; temporal measurement; tool

Abstract Cellular mechano-transduction is important for detecting environmental changes and Piezo channels, which were discovered in 2010, have crucial roles in several processes in mammalian cells, such as vascular development and function, pulmonary respiration, and sensory transduction. Their conservation in unicellular organisms and their function in Ca2+ signaling is an untapped area of research. Ca2+ is a potent signal for controlling a variety of downstream effectors that stimulate specific biological responses. We and others discovered years ago that contact with host cells triggers an increase in cytosolic Ca2+ of the infective trypomastigote stage of Trypanosoma cruzi, the etiologic agent of Chagas disease. Preventing this Ca2+ increase with intracellular Ca2+ chelators prevented host cell invasion. A role for Ca2+ in T. cruzi replication and differentiation was also proposed. The mechanism involved in the Ca2+ increase upon contact of trypomastigotes with host cells was never identified but its contact-dependence suggests a mechano-transduction process. Stimulation of Piezo channels preferentially leads to Ca2+ and in some cases Na+ and Ca2+ entry into cells. Two paralogs, TcPiezo1 and TcPiezo2, are present in T. cruzi. Using CRISPR/Cas9 techniques we have C-terminally tagged TcPiezo1 and localized it to the T. cruzi plasma membrane and an intracellular spot. We also obtained TcPiezo1-KO mutants, which are deficient in host cell invasion and intracellular replication. We propose to study the role of both Piezo channels in Ca2+ signaling during T. cruzi host cell invasion and replication. Our hypothesis is that T. cruzi Piezo channels will function at the plasma membrane as Ca2+ entry channels that will activate a signaling cascade culminating in specific parasite functions like activation of invasion and differentiation. With the aim of studying cytosolic Ca2+ concentration fluctuations in the infective stages of T. cruzi we propose to use genetically encoded Ca2+ indicators (GECIs), in addition to the traditional chemical indicators. GECIs are powerful tools that allow the noninvasive imaging of defined cells and compartments. The use of these new tools will facilitate direct real-time observation of Ca2+ changes during T. cruzi host cell invasion and replication, the investigation of the role of Piezo channels in the generation of these changes, and the study of other potential Ca2+ roles in future work. We propose to generate T. cruzi different stages expressing GECIs targeted to their cytosol and to their mitochondria to study the dynamics of Ca2+ in live parasites and explore the requirement for Ca2+ signaling during host cell invasion by trypomastigotes and during replication and differentiation of amastigotes, and the role of Piezo channels in these processes. T. cruzi Piezo channels will likely have lineage specific characteristics, which are different from those of mammalian cells Piezo channels, and their study could lead to new insights into the T. cruzi biology and the potential identification of new targets.

摘要 细胞机械转导对于检测环境变化和Piezo通道是重要的， 它们于2010年被发现，在哺乳动物细胞的几个过程中起着至关重要的作用，例如 血管发育和功能、肺呼吸和感觉传导。它们的保护在 单细胞生物及其在钙信号转导中的作用是一个尚未开发的研究领域。Ca2+是一种强效的 用于控制刺激特定生物反应的各种下游效应器的信号。我们和 其他人几年前就发现，与宿主细胞接触会触发细胞内钙离子的增加 查加斯病的病原体克氏锥虫感染性锥虫鞭毛虫期。预防 这种与细胞内钙离子螯合剂一起增加的钙离子可以防止宿主细胞的入侵。钙离子在克氏锥虫中的作用 并提出了复制和分化的建议。细胞内钙离子升高的机制 类鞭毛虫与宿主细胞的接触从未被鉴定过，但它的接触依赖性表明 机械转导过程。对Piezo通道的刺激优先导致钙离子，在某些情况下 3例Na+、Ca~(2+)进入细胞。TcPiezo1和TcPiezo2是克氏毛滴虫的两个对虾。vbl.使用 CRISPR/Cas9技术我们用C-末端标记TcPiezo1并将其定位于克氏锥虫血浆 细胞膜和细胞内的斑点。我们还获得了宿主缺乏的TcPiezo1-KO突变体 细胞侵袭和细胞内复制。我们建议研究两种Piezo通道在钙离子中的作用 克氏锥虫宿主细胞入侵和复制过程中的信号转导。我们的假设是，克氏锥虫的Piezo通道 将在质膜上作为钙离子进入通道发挥作用，激活信号级联 最终导致特定的寄生虫功能，如激活入侵和分化。目的是为了 我们拟用来研究克氏毛滴虫感染阶段细胞内钙离子浓度的变化 除了传统的化学指示剂外，还有遗传编码的钙指示剂(GECI)。GECI是 强大的工具，允许对已定义的细胞和隔室进行非侵入性成像。这些新技术的使用 这些工具将有助于直接实时观察克氏锥虫宿主细胞入侵和感染期间钙离子的变化 复制，研究压电通道在这些变化的产生中的作用，以及这项研究 关于未来工作中其他潜在的钙离子作用。我们提出了产生T.ruzi的不同阶段表达 GECI定位于它们的胞浆和线粒体，以研究活体寄生虫和 探讨锥虫侵入宿主细胞过程中对钙信号的需求 无鞭毛体的复制和分化，以及Piezo通道在这些过程中的作用。克氏锥虫 压电通道将可能具有特定于世系的特征，这与 哺乳动物细胞Piezo通道，他们的研究可能导致对T.ruzi生物学和 新目标的潜在识别。