Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

Dutta, Soumita; Avasthi, Prachee

doi:10.1128/msphere.00003-17

Cited by 3 publications

(1 citation statement)

References 71 publications

(76 reference statements)

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“…Replication of its cilium is an essential part of T. brucei ’s cell cycle [40]. Basal body duplication, followed by nuclear and kinetoplast DNA replication, initiates cilium replication [41].…”

Section: Resultsmentioning

confidence: 99%

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei

Godar

Oristian

Hinsch

et al. 2021

Preprint

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Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids, including trypanosomes. Trypanosoma brucei flagella exhibit a bending wave that propagates from the flagellum’s tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein-associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex-type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii . However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi, rescued the knockdown with eGFP-tagged TbLC2, and quantified TbLC2’s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown resulted in kinetoplast mislocalization and the formation of multiple cell clusters in cell culture. We also found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to-tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids' unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.

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Section: Resultsmentioning

confidence: 99%

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei

Godar

Oristian

Hinsch

et al. 2021

Preprint

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Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii

Jack

Avasthi

2018

Methods in Molecular Biology

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Flagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of vertebrate cells and provide an excellent model to study ciliogenesis. Cilia and flagella are important organelles used for motility and sensing the extracellular environment. Abnormalities in cilia structure or ciliary dysfunction can have devastating consequences ranging from diabetes and obesity to polycystic kidney disease and mental retardation. Small-molecule inhibitor libraries can be used to screen for flagellum-associated phenotypes in assembly, length, motility, deflagellation, and cellular toxicity. These phenotypes can be assessed from direct microscopic visualization and custom-designed assays. These methods identify fundamental regulators of ciliary biology as well as potential therapeutic interventions for ciliopathies.

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Cell-based biosynthesis of linear protein nanoarrays

Ishikawa

Tian

et al. 2019

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI

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Flagellar Synchronization Is a Simple Alternative to Cell Cycle Synchronization for Ciliary and Flagellar Studies

Cited by 3 publications

References 71 publications

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei

Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei

Chemical Screening for Flagella-Associated Phenotypes in Chlamydomonas reinhardtii

Cell-based biosynthesis of linear protein nanoarrays

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