{"title":"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。","authors":"Heidi Anderson, Rhonda Reigers Powell, Meredith Teilhet Morris","doi":"10.1111/jmi.70019","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":16484,"journal":{"name":"Journal of microscopy","volume":" ","pages":""},"PeriodicalIF":1.9000,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Ultrastructural expansion microscopy reveals unexpected levels of glycosome heterogeneity in African trypanosomes.\",\"authors\":\"Heidi Anderson, Rhonda Reigers Powell, Meredith Teilhet Morris\",\"doi\":\"10.1111/jmi.70019\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":16484,\"journal\":{\"name\":\"Journal of microscopy\",\"volume\":\" \",\"pages\":\"\"},\"PeriodicalIF\":1.9000,\"publicationDate\":\"2025-07-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of microscopy\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1111/jmi.70019\",\"RegionNum\":4,\"RegionCategory\":\"工程技术\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MICROSCOPY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of microscopy","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1111/jmi.70019","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MICROSCOPY","Score":null,"Total":0}
Ultrastructural expansion microscopy reveals unexpected levels of glycosome heterogeneity in African trypanosomes.
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.
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.