A fixable probe for visualizing flagella and plasma membranes of the African trypanosome

PloS One
Justin Wiedeman, Kojo Mensa-Wilmot

Abstract

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) destin...Continue Reading

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Citations

Jun 18, 2019·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Kamila CzechowskaAttila Tárnok
Jan 16, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Shuo ZhangMark Bradley
Oct 23, 2018·ACS Medicinal Chemistry Letters·Jennifer L WoodringMichael P Pollastri

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Methods Mentioned

BETA
fluorescence microscopy
super-resolution
super-resolution microscopy
flow cytometry
light microscopy
confocal microscopy

Software Mentioned

Simple Neurite Tracer ( SNT )
IDEAS
SIM
SNT
Prism
Fiji
GraphPad
Neurite Tracer
mCLING
SR

Related Concepts

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African Trypanosomiasis

African trypanosomiasis, also known as sleeping sickness, is an insect-borne parasitic disease of humans and other animals. It is caused by protozoa of the species Trypanosoma brucei and almost invariably progresses to death unless treated. Discover the latest research on African trypanosomiasis here.