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Intraflagellar transport (IFT) sculpts the proteome of cilia and flagella; the antenna-like organelles found on the surface of virtually all human cell types. By delivering proteins to the growing ciliary tip, recycling turnover products, and selectively transporting signalling molecules, IFT has critical roles in cilia biogenesis, quality control, and signal transduction. IFT involves long polymeric arrays, termed IFT trains, which move to and from the ciliary tip under the power of the microtubule-based motor proteins kinesin-II and dynein-2. Recent top-down and bottom-up structural biology approaches are converging on the molecular architecture of the IFT train machinery. Here we review these studies, with a focus on how kinesin-II and dynein-2 assemble, attach to IFT trains, and undergo precise regulation to mediate bidirectional transport.

Cytoplasmic dynein-2 is a motor protein complex that drives the movement of cargoes along microtubules within cilia, facilitating the assembly of these organelles on the surface of nearly all mammalian cells. Dynein-2 is crucial for ciliary function, as evidenced by deleterious mutations in patients with skeletal abnormalities. Long-standing questions include how the dynein-2 complex is assembled, regulated, and switched between active and inactive states. A combination of model organisms, in vitro cell biology, live-cell imaging, structural biology and biochemistry has advanced our understanding of the dynein-2 motor. In this Cell Science at a Glance article and the accompanying poster, we discuss the current understanding of dynein-2 and its roles in ciliary assembly and function.

Trypanosomes of the genus Leishmania swim by undulating motions of a single flagellum driven by axonemal dynein ATPases, essential for parasite survival and infectivity. The flagellum possesses two waveforms; flagellar (tip-to-base) responsible for forward movements and ciliary (base-to-tip) possibly responsible for reorientation in response to changes in surroundings. However, the role of dyneins in regulating the two waveforms remains unknown. Moreover, the unpredictable nature of the parasite ciliary waveform makes it difficult to study. We have previously reported a detergent-extracted, ATP-reactivated model ideal for investigating flagellar motility regulation in Leishmania that allows one to generate reactivated Leishmania flagella with constitutively beating ciliary waves in presence of cyclic-AMP. Here, using three dynein inhibitors [erythro-9-(2-hydroxy-3-nonyl) adenine, ciliobrevin A and vanadate] we investigated the role of dyneins in regulating the two waveforms of Leishmania. Using high speed videomicroscopy we observed differential inhibition of beat frequencies and waveforms of flagellar and ciliary beats in live (in vivo) and ATP-reactivated (in vitro) parasites. Beat frequency of flagellar waveform was more strongly reduced than ciliary waveform. Surprisingly, inhibition of the ciliary waveform led to an altered phenotype with the distal half of the flagellum paralysed. ATPase assays confirmed that dynein activity of flagellar cells was more strongly inhibited compared to ciliary cells irrespective of the mechanism of inhibition. Possibly the two different waveforms are an outcome of changes in the mechanical properties of axonemal dyneins present at the tip of the flagellum that contains a sliding resistive structure. Our study suggests that dyneins responsible for the two waveforms in Leishmania bear different structural and functional conformations. Moreover, during ciliary beating, there is heterogeneity along the flagellum.

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni . However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani .

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250μM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes.

Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60-WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60-WDR34-light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.

Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania. However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena, the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp., Highlights • Identified outer and inner-arm dynein heavy chain genes in five Leishmania species. • Outer-arm dyneins of the genus Leishmania are two-headed like metazoans. • Conserved IQ-like motif present in outer-arm α and inner-arm 1α in Leishmania sp. • Conserved serine and threonine residues in dynein arms exclusive to Leishmania sp. • Possible regulation of dynein activity via phosphorylation of these IQ-like motifs.

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.