Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella

Abstract

Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults

“…IFT particles are often visible by EM as electron-dense ‘IFT trains’ located between the MTs and the ciliary membrane (Rogowski et al, 2013; Stepanek and Pigino, 2016; Vannuccini et al, 2016). Interestingly, in our EM analysis of WT cilia we could not find such particles localised between the B MTs and the ciliary membrane in the axoneme, but we instead found similar extended electron-dense particles localising along the A MTs and facing the internal lumen of the BB–TZ junction (arrows, Fig.…”

The Intraflagellar Transport Machinery

“…A structural building block (most likely corresponding to the IFT complex and associated cargos) was seen to form a dimer that repeated every 40 nm along the longitudinal axis of the flagellum, implying that the IFT complexes not only make longitudinal but also lateral contacts (Pigino et al 2009). While it was initially speculated that these long assemblies represented IFT trains moving in the anterograde direction (Pigino et al 2009), it now became clear that they, in fact, are stalled trains, and that both anterograde and retrograde trains are significantly shorter and display a different periodicity (Stepanek and Pigino 2016;Vannuccini et al 2016). The molecular basis of IFT train formation is still not understood, and it is not clear whether the IFT complex itself is able to form trains or whether association with motors, cargos, or other accessory factors is required.…”

Intraflagellar transport: mechanisms of motor action, cooperation, and cargo delivery

“…The authors hypothesized that the selection of B-versus A-tubules by trains moving in opposite directions may avoid collisions between them, although we note that this could also be accomplished by the selective use of the 10-15 PF tracks that are available per singlet or through the use of distinct A-tubules [83], and that collisions do not seem to pose a significant problem for IFT trains that move bidirectionally along the DS A-tubules of C. elegans cilia [78]. The precise correlation between the morphologically defined IFT trains visible by EM and the biochemically defined protein components of the IFT machinery (discussed next) is currently an outstanding unresolved problem in the field of cilium biology [81].…”