Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, United Kingdom.
Department of Pathobiology and Population Sciences, The Royal Veterinary College, University of London, North Mymms, United Kingdom.
Front Cell Infect Microbiol. 2023 Mar 9;13:1082622. doi: 10.3389/fcimb.2023.1082622. eCollection 2023.
Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of , parasitic coccidian protozoa.
High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells.
Live cell imaging of MDBK cells infected with sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape.
MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.
折光体（RB）是一种未知功能的大型无膜细胞器（MLO），在所有种属的疟原虫寄生性顶复门原虫的孢子虫阶段，作为一对突出的不匹配体存在。
使用高分辨率成像方法，包括延时活共聚焦显微镜和连续块面扫描电子显微镜（SBF-SEM），在孢子虫侵入宿主细胞前后研究 RB 和其他细胞内细胞器的形态。
用 MDBK 细胞感染 孢子虫的活细胞成像证实了以前的报道，即 RB 在感染后从两个减少到一个，并且表明 RB 数量的减少发生在前 RB 与后 RB 的融合，这一过程持续 20-40 秒，发生在感染后 2-5 小时之间。使用 SBF-SEM 对整个单个孢子虫进行超微结构研究，包括在入侵宿主细胞前后，证实了活细胞成像观察结果，并表明 RB 融合伴随着整个孢子虫细胞形状的变化。此外，融合后的单个 RB 体积比融合前的两个 RB 更大。使用肌动蛋白抑制剂研究肌动蛋白在 RB 融合中的潜在作用，细胞松弛素 D 显著抑制 RB 融合和伴随的孢子虫细胞形状变化。
真核生物中的 MLO 以缺乏膜和能够进行液-液相分离（LLPS）和融合为特征，通常以肌动蛋白介导的方式进行。基于在 RB 融合时观察到的孢子虫细胞形状的变化，以及肌动蛋白在这个过程中的潜在作用，我们提出 RB 被归类为 MLO，并被认为是迄今为止所描述的最大的 MLO 之一。
