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Abstract
The flagellum of parasitic trypanosomes is a multifunctional appendage essential for its viability and infectivity. However, the biological mechanisms that make the flagellum so dynamic remains unexplored. No method is available to access and induce axonemal motility at will to decipher motility regulation in trypanosomes. For the first time we report the development of a detergent-extracted/demembranated ATP-reactivated model for studying flagellar motility in Leishmania. Flagellar beat parameters of reactivated parasites were similar to live ones. Using this model we discovered that cAMP (both exogenous and endogenous) induced flagellar wave reversal to a ciliary waveform in reactivated parasites via cAMP-dependent protein kinase A. The effect was reversible and highly specific. Such an effect of cAMP on the flagellar waveform has never been observed before in any organism. Flagellar wave reversal allows parasites to change direction of swimming. Our findings suggest a possible cAMP-dependent mechanism by which Leishmania responds to its surrounding microenvironment, necessary for its survival. Our demembranated-reactivated model not only serves as an important tool for functional studies of flagellated eukaryotic parasites but has the potential to understand ciliary motility regulation with possible implication on human ciliopathies.
Highlights
Most of our current understanding of the regulatory mechanism controlling flagellar and ciliary beating come from detergent-extracted/demembranated, ATP reactivation studies in organisms like sperms of sea urchins, Ciona, dog, bull, flagella of Chlamydomonas and cilia of Paramecium[13,14,15,16,17,18]
The absence of reports elucidating the control mechanism of flagellar motility in Leishmania led us to questions like: can the demembranated Leishmania flagella be reactivated? If so would it be possible to tease out the regulatory mechanisms of flagellar motility? We presume that such a reactivated model will allow numerous studies in the future elucidating leishmanial flagellar motility and associated functionality for its survival and infection
Based on the conditions that led to improved beat frequency, we developed the following protocol namely Leishmania Reactivation Protocol (LRP)
Summary
Most of our current understanding of the regulatory mechanism controlling flagellar and ciliary beating come from detergent-extracted/demembranated, ATP reactivation studies in organisms like sperms of sea urchins, Ciona, dog, bull, flagella of Chlamydomonas and cilia of Paramecium[13,14,15,16,17,18]. We presume that such a reactivated model will allow numerous studies in the future elucidating leishmanial flagellar motility and associated functionality for its survival and infection. The absence of reports elucidating the control mechanism of flagellar motility in Leishmania led us to questions like: can the demembranated Leishmania flagella be reactivated? Such a model would have the potential to study the signalling pathways that possibly malfunction in ciliopathies
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