Date Published: May 16, 2018
Publisher: Public Library of Science
Author(s): Justin Wiedeman, Kojo Mensa-Wilmot, Marek Cebecauer.
The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.
Trypanosoma brucei is a protozoan that causes Human African Trypanosomiasis and nagana in cattle (reviewed in [1–2]). T. brucei has been the subject of many studies to understand unique aspects of its biology (e.g. antigenic variation, RNA editing, trans-splicing, and catenated mitochondrial DNA). Fluorescence microscopy is frequently used to visualize cells, localize trypanosome proteins , and detect small molecule drugs intracellularly . Resolution of the technique is constrained by the diffraction limit of visible light, as it cannot distinguish fluorescent objects within ≈200 nm lateral to each other (reviewed in ). Super-resolution fluorescence microscopy techniques, such as structured illumination and stimulated emission depletion (STED) enable resolution of objects within 200 nm lateral to each other [5–6], and have been used to study T. brucei [7,8]. For a cell as small as a trypanosome (≈2 μm wide, ≈25 μm long), super-resolution microscopy offers notable advantages over standard fluorescence microscopy.
Fluorescence microscopy is an important tool for trypanosome cell biology , and recent technical advancements have enabled the visualization of objects smaller than the resolution limit imposed by diffraction of light. In trypanosome biology, few “fixable”, fluorescent, non-reactive probes for labeling the plasma membrane of bloodstream form T. brucei are available , and these are not known to be compatible in common super-resolution techniques that have specific fluorophore requirements . We have attempted to fill this gap in fluorescence microscopy of bloodstream form trypanosomes. Using 3D SR-SIM, we demonstrate that the lipopeptide probe mCLING labels plasma and flagellar membranes, and endocytic stations. Further, mCLING is fixable and can be used for immunofluorescence microscopy.