Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry

Abstract

The 9+2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base (and may be symmetrical or asymmetrical. We hypothesised that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-tobase or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction. Significance statementThe motile flagellum/cilium is found across all eukaryotic life, and it performs critical functions in many organisms including humans. A fundamental requirement for a motile flagellum/cilium is that it must undergo the correct and appropriate waveform for its specific function. Much is known about the generation of asymmetry in flagellum movement, however it is unknown how a motile flagellum specifies where waves should start and whether waves should go from base-to-tip, or from tip-tobase. We show here that in two flagellum model organisms (the human parasites Trypanosoma brucei and Leishmania mexicana, differences in the outer dynein arms between the distal and proximal regions of the flagellum determine wave propagation direction, and are generated and maintained by the flagellum growth machinery.

Dealing with several flagella in the same cell

“…This could be related to its function because this protein is required for the assembly of outer dynein arms at the distal portion of the flagellum (Bonnefoy et al, 2018) that is characterised by a unique set of dynein arm docking complex (Edwards et al, 2018). This has been observed with the ODA8/LRRC56 protein in T. brucei that is mostly present in growing flagella but not in flagella undergoing maintenance.…”

“…This has been observed with the ODA8/LRRC56 protein in T. brucei that is mostly present in growing flagella but not in flagella undergoing maintenance. This could be related to its function because this protein is required for the assembly of outer dynein arms at the distal portion of the flagellum (Bonnefoy et al, 2018) that is characterised by a unique set of dynein arm docking complex (Edwards et al, 2018).…”

The single flagellum ofLeishmaniahas a fixed polarisation of its asymmetric beat

“…In Chlamydomonas, regulation of the symmetric dynamic curvature of the flagellum to make a wave 298 propagate and the static curvature of the flagellum to introduce asymmetry are separable, and some 299 mutants (such as mbo2) cannot form a static curvature giving an aberrant symmetric waveform 300 (Brokaw and Kamiya, 1987;Geyer et al, 2016;Segal et al, 1984). In Leishmania, no mutants are yet 301 known which lose the asymmetry of their base-to-tip beat -deletions of the outer dynein arm-302 associated proteins dDC2 and LC4-like do promote asymmetric or symmetric beats respectively, but 303 these beats occur in their normal base-to-tip and tip-to-base propagation directions respectively 304 (Edwards et al, 2018). However, Leishmania deletion mutants (including PF16, Hydin, IC140) are 305 known where the flagellum is paralysed but often still has a static curvature (Beneke et al, 2019) 306 while, naïvely, paralysed flagella would be expected to be straight.…”

“…Together, this work greatly constrains the molecular origin of asymmetry in Leishmania base-to-tip 322 beats. Our previous work indicated outer dynein arm-associated factors are likely important for 323 switching between symmetric tip-to-base and asymmetric base-to-tip beats (Edwards et al, 2018), 324 however controlling switching is distinct from the actual generation of asymmetry. The most likely 325 remaining candidate is differences between the outer doublet decorations, in particular in the region 326 of inner arm dynein b based on cryo-electron tomography of T. brucei (Imhof et al, 2019).…”

“…For endogenous tagging of L. mexicana genes, constructs and sgRNAs were generated using the PCR-338 based approaches previously described (Beneke et al, 2017;Dean et al, 2015), using the pLPOT (also 339 called pLrPOT) (Edwards et al, 2018) series of plasmids as the PCR template. L. mexicana were 340 transfected and subjected to drug selection as previously described (Dean et al, 2015).…”