Ultrastructural expansion microscopy reveals unexpected levels of glycosome heterogeneity in African trypanosomes.

IF 1.9 4区 工程技术 Q3 MICROSCOPY
Heidi Anderson, Rhonda Reigers Powell, Meredith Teilhet Morris
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引用次数: 0

Abstract

Kinetoplastid parasites include several species. Trypanosoma brucei causes African sleeping sickness in humans and a wasting disease nagana in livestock. Trypanosoma cruzi is the causative agent of Chagas disease and Leishmania species cause leishmaniasis, which can present with visceral, cutaneous, or mucocutaneous symptoms. All kinetoplastids harbour specialised peroxisomes called glycosomes, so named because most of the glycolytic pathway that is cytosolic in other eukaryotes is localised to these organelles. Glycosomes lack DNA and are essential for parasite viability. Despite their name, glycosomes also house enzymes involved in diverse pathways, including the pentose phosphate pathway, ether lipid biosynthesis, purine salvage, and sugar nucleotide biosynthesis. The degree to which these biochemical pathways localise together within the same organelle or to different glycosome populations is unclear. Biochemical fractionations and imaging data strongly suggest that glycosomes are heterogeneous in composition and that even within a single parasite, there are different glycosome populations. Until recently, we lacked the technology to systematically characterise glycosome populations within parasites. Glycosome morphology, composition, and localisation have historically been studied using widefield fluorescence and electron microscopy (EM). While EM can resolve individual organelles, it is extremely low throughput and requires specialised expertise and equipment. Widefield fluorescence imaging is higher throughput and more accessible. However, the small size of T. brucei cells, which are ∼20 µM in length and 3-5 µM in width, and glycosomes (100 nm in diameter) place these organelles below the resolution limits of standard microscopy and require super-resolution techniques to be resolved. These resolution issues are compounded by the cytoplasm's crowded nature, making it hard to discern individual organelles from each other. To overcome this, we leveraged recent advances in super-resolution microscopy, including a method called Ultrastructure Expansion Microscopy (U-ExM) combined with confocal imaging and LIGHTNING™ deconvolution to optimise the resolution of individual glycosomes. We found that antibodies against two different glycosome marker proteins (aldolase and GAPDH) exhibit discrete staining patterns. This high-resolution approach also revealed that glycosome morphology varies between monomorphic parasites that cannot complete the lifecycle and pleomorphic parasites that can, and is dynamically influenced by extracellular conditions, such as glucose availability, underscoring the adaptability of T. brucei's compartmentalisation to environmental changes.

超微结构扩展显微镜显示非洲锥虫的糖体异质性出乎意料。
着丝质体寄生虫包括几种。布鲁氏锥虫在人类中引起非洲昏睡病,在牲畜中引起那格那病。克氏锥虫是恰加斯病的病原体,利什曼原虫引起利什曼病,可出现内脏、皮肤或粘膜皮肤症状。所有的着丝质体都含有特殊的过氧化物酶体,称为糖体,之所以如此命名,是因为其他真核生物的细胞质中的大多数糖酵解途径都定位于这些细胞器。糖体缺乏DNA,对寄生虫的生存至关重要。尽管它们的名字是糖体,但糖体也容纳了参与多种途径的酶,包括戊糖磷酸途径、醚脂生物合成、嘌呤回收和糖核苷酸生物合成。这些生化途径在同一细胞器或不同糖体群体中共同定位的程度尚不清楚。生化分离和成像数据强烈表明,糖体在组成上是不均匀的,即使在一个寄生虫内,也存在不同的糖体种群。直到最近,我们还缺乏系统地表征寄生虫体内糖体种群的技术。糖体的形态,组成和定位历史上研究使用宽视场荧光和电子显微镜(EM)。虽然EM可以分解单个细胞器,但通量极低,需要专门的专业知识和设备。宽视场荧光成像是更高的吞吐量和更容易获得。然而,布鲁氏虾细胞的小尺寸(长约20µM,宽3-5µM)和糖体(直径100 nm)使这些细胞器低于标准显微镜的分辨率限制,需要超分辨率技术来解决。这些分辨率问题与细胞质拥挤的性质相结合,使得很难区分单个细胞器。为了克服这个问题,我们利用了超分辨率显微镜的最新进展,包括一种称为超结构扩展显微镜(U-ExM)的方法,结合共聚焦成像和LIGHTNING™反卷积来优化单个糖体的分辨率。我们发现针对两种不同糖体标记蛋白(醛dolase和GAPDH)的抗体表现出离散的染色模式。这种高分辨率的方法还揭示了糖体形态在不能完成生命周期的单形态寄生虫和可以并受细胞外条件(如葡萄糖可用性)动态影响的多形性寄生虫之间存在差异,强调了布氏体的区隔化对环境变化的适应性。
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来源期刊
Journal of microscopy
Journal of microscopy 工程技术-显微镜技术
CiteScore
4.30
自引率
5.00%
发文量
83
审稿时长
1 months
期刊介绍: The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit. The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens. Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.
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