University of Konstanz, Konstanz, Germany.
J Eukaryot Microbiol. 2022 Sep;69(5):e12895. doi: 10.1111/jeu.12895. Epub 2022 Mar 7.
A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca -regulating proteins, including Ca -channels, Ca -pumps, Ca -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca -influx channels, locally enriched intracellular InsP -type (InsP R, mainly in osmoregulatory system) and ryanodine receptor-like Ca -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca -influx, that is, a mechanism called "store-operated Ca -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca -concentration, [Ca ] , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.
草履虫细胞的膜相互作用类型与哺乳动物细胞一样多，这是 R. Allen 和 A. Fok 用单克隆抗体确定的。从那时起，我们已经在分子水平上确定了关键参与者，如 SNARE 蛋白、Ca 调节蛋白，包括 Ca 通道、Ca 泵、不同亲和力的 Ca 结合蛋白等，研究了它们的功能，并在光镜和电镜水平上对其进行了定位。SNARE 蛋白与类似于突触结合蛋白的 Ca 传感器蛋白一起介导膜融合。这种相互作用还受到单体 GTP 酶的调节，A. Turkewitz 已经确定了四膜虫和草履虫中的 GTP 酶谱。与哺乳动物细胞一样，GTP 酶在与 H+-ATP 酶共同作用下，通过腔内酸化而在膜上被激活。对于这些复杂的分子，我们在草履虫中发现了数量惊人的 17 个 a 亚基同工酶，它们连接着聚合物头部和基础部分，V1 和 V0。（这种多样性可能反映了不同的局部功能需求。）与质膜 Ca 内流通道一起，局部富集的细胞内 InsP 型（InsP R，主要在渗透压调节系统中）和 Ryanodine 受体样 Ca 释放通道（Ryanodine 受体样蛋白，RyR-LP）一起，这种复杂性介导了大多数灵活的局部膜到膜相互作用的 Ca 信号。正如我们所发现的，后两种通道类型缺少相当一部分 N 端部分。咖啡因和 4-氯间甲酚（在恶性失眠症患者手术中用于探测 Ryanodine 受体突变的药物）通过激活肺泡囊中周边部分的 Ca 释放通道 CRC-IV 引发纤毛囊释放。这是由 Ca 内流叠加的，即所谓的“储存操纵的 Ca 内流”（SOCE）。对于大多数关键参与者，我们已经在整个草履虫细胞中绘制了同工酶图谱，这些同工酶在参与囊泡运输的不同细胞器中具有共同或不同的特征。游离 Ca 浓度的局部值[Ca]及其变化，例如，在 exocytosis 刺激时，已通过荧光染料和螯合剂效应进行了记录。同时，我们通过与冷冻固定和 X 射线微分析相结合的猝灭流动分析记录了肺泡囊中 Ca 的释放。
