Abstract
ABSTRACTAnalysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be extremely powerful. Here, high-speed multifocal plane fluorescence microscopy, where the light path is split to visualise multiple focal planes simultaneously, was used to reconstruct Trypanosoma brucei and Leishmania mexicana movement in 3D. These species are uniflagellate unicellular parasites for which motility is vital. It was possible to use either a fluorescent stain or a genetically-encoded fluorescent protein to visualise flagellum and cell movement at 200 Hz frame rates. This addressed two open questions regarding Trypanosoma and Leishmania flagellum beating, which contributes to their swimming behaviours: 1) how planar is the L. mexicana flagellum beat, and 2) what is the nature of flagellum beating during T. brucei ‘tumbling’? We showed that L. mexicana has notable deviations from a planar flagellum beat, and that during tumbling the T. brucei flagellum bends the cell and beats only in the distal portion to achieve cell reorientation. This demonstrates high-speed multifocal plane fluorescence microscopy as a powerful tool for the analysis of beating flagella.
Highlights
Trypoanosomatid parasites, including the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., have a single flagellum whose motility is vital for progression through the life cycle (Beneke et al, 2019; Broadhead et al, 2006; Rotureau et al, 2013; Shimogawa et al, 2018)
Multifocal plane imaging was initially tested with T. brucei labelled with FM 4-64FX in normal growth medium
This work demonstrates that multifocal plane fluorescence microscopy is capable of analysing volumes and frame rates useful for interrogating flagellum beating using either chemical or native fluorescent protein fluorescence
Summary
Trypoanosomatid parasites, including the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., have a single flagellum whose motility is vital for progression through the life cycle (Beneke et al, 2019; Broadhead et al, 2006; Rotureau et al, 2013; Shimogawa et al, 2018). Trypanosoma brucei trypomastigotes, including procyclic (fly midgut) and mammalian bloodstream forms, have a complex threedimensional (3D) cell movement (Bargul et al, 2016; Heddergott et al, 2012; Rodríguez et al, 2009; Schuster et al, 2017; Wheeler, 2017) They can swim in liquid, but the fly gut and salivary glands. Received 10 March 2019; Accepted 22 July 2019 can be densely packed with parasite cells (Schuster et al, 2017) When they are free in a volume of liquid, such as the bloodstream, they swim far slower than the fluid flow (Heddergott et al, 2012; Krüger et al, 2018), yet they can invade tissues (Capewell et al, 2016; Trindade et al, 2016). As for T. brucei, they will probably undergo hydrodynamic coupling when densely packed, which could give collective population movement at the macro scale
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
