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

Abstract

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.

LeishIF3d is a non-canonical cap-binding protein in Leishmania

“…The morphological changes observed for the LeishIF3d(+/−) cells are in accordance with the reduced expression of the dynein light chain (LmxM.13.1180), and Kinesin (LmxM.17.1110). These regulate ciliary movement in Trypanosoma brucei (Godar et al, 2022) and are essential for cell morphogenesis, cell division, and virulence (Corrales et al, 2021). Reduced expression was recorded also for proteins that encode a variety of metabolic enzymes.…”

Long-range electrostatic interactions significantly modulate the affinity of dynein for microtubules

“…We performed force-dependent binding affinity assays using an optical tweezers with a 1064 nm, 10 W ytterbium fiber laser (YLR-10-1064-LP, IPG Photonics, Oxford, MA) focused on the sample plane by an oil immersion objective (CFI60 Plan Apochromat Lambda 60Â N.A. 1.4, Nikon Instruments, Melville, NY), as described previously (48). Multiple polarizing beam splitters, zero-order half-wave plates, beam traps, and neutral optical density filters (ThorLabs, Newton, NJ) direct, condition, and regulate the laser power at the sample plane.…”