Abstract
Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.
Highlights
Kinetoplastida are a group of protists, comprising both free-living and parasitic organisms
We employed a variation of Expansion microscopy (ExM) termed ultrastructure ExM, which is based on fixation of cells with 4% formaldehyde and 4% acrylamide, followed by gelation with 19% sodium acrylate, 10% acrylamide and 0.1% N, N’-methylenebisacrylamide, denaturation with 200 mM sodium dodecyl sulfate at 95°C, and antibody staining [10]
We observed that ExM is capable of faithfully reproducing information on the presence, positioning, and morphology of structures and structural components
Summary
Kinetoplastida are a group of protists, comprising both free-living and parasitic organisms. The most notorious are Trypanosoma brucei (the causative agent of African sleeping sickness in humans and nagana in cattle), T. cruzi (causing Chagas disease in Americas) and numerous species of Leishmania genus (causing cutaneous, mucocutaneous and visceral leishmaniosis on several continents) These important parasites are transmitted between mammalian hosts by insect vectors. Despite the considerable morphological diversity, kinetoplastid cells share a common design principle with their morphology being determined by the microtubule-based cytoskeleton; the microtubules form the subpellicular corset, an array of parallel evenly spaced microtubules, which subtend the entire cytoplasmic membrane [2] Another prominent feature is the presence of the flagellum, the principal motility organelle, which contains the evolutionarily conserved microtubulebased axoneme, and the kinetoplastid-specific paraflagellar rod [2]. Kinetoplastid cells of a proliferative population show a remarkable uniformity of their cytoskeletal architecture
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