Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

Abstract

The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.transition zone | cilium/flagellum | BBSome | MKS/B9 complex | trypanosome C ilia, or flagella (the two terms are used here interchangeably), are multifunctional organelles that were present in the last common eukaryotic ancestor (1). Defects in cilia are responsible for pleiotropic human diseases (called ciliopathies) and their function is essential for pathogenesis in protozoan parasites (2, 3).During ciliogenesis, a microtubule organizing center called the "basal body" docks at the membrane. Basal bodies are built from nine microtubule triplets and two microtubules from each triplet extend to form a transition zone (TZ) and, ultimately, the axoneme. Thus, the TZ represents a structural junction between the basal body and the axoneme.As expected from its strategic position between the basal body and the axoneme, the TZ acts as a "ciliary gate" that controls ciliary composition and function (4). Many ciliopathies are caused by defects in complexes that associate with the TZ, including Meckel syndrome (MKS), Joubert syndrome and Bardet-Biedl syndrome (BBS). Studies using murine kidney epithelial cells and embryos suggest that the MKS complex demarcates the ciliary membrane by forming a diffusion barrier at the base of the cilium (5-7). On the other hand, the BBS complex (BBSome) is an adapter for intraflagellar transport (IFT) complexes that cross the TZ barrier to transport sensory proteins between the ciliary membrane and the cell body (8, 9).The TZ has distinctive structural features. At the proximal boundary of the TZ, where the basal body C tubule terminates, a "terminal plate" crosses the TZ. Studies in Tetrahymena suggest that the terminal plate contains pores for the passage of IFT "trains" that deliver axonemal components to the distal tip of flagella (10). Striated transitional fibers radiate from the distal end of the basal body triplets to join the plasma membrane (11-14), forming blades thought to create a physical barrier preventing vesicles from entering the ciliary lumen. Electron microscopy (EM) studies in Chlamydomonas suggest that...

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

“…Washed cells were settled on glass slides then immediately observed. To generate cytoskeletons, cells were prepared as for live cell microscopy, the membrane solubilised with 0.5% NP40 in PEME (100 mM PIPES•NaOH pH 6.9, 2 mM EGTA, 1 mM MgSO 4 We could not detect the expected blue fluorescence of the Coumarin ligand, but found it was an effective block, as described previously (67). Widefield epifluoresence and phase contrast images were captured using a Zeiss Axioimager.Z2 microscope with a 63× NA 1.40 oil immersion objective and a Hamamatsu ORCA-Flash4.0 camera.…”

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

“…Moreover, the majority of these proteins were not detected in previous flagellar fractions; hence their substantial enrichment before MS analysis appears fundamental for their detection. We recently used a similar approach for identification of constituents of the trypanosome TZ (30). Hence, the SIP approach is highly successful for analyzing discrete protein structures and regions of the cytoskeleton of cells.…”

Dealing with several flagella in the same cell

“…At the very base of the TZ, transition fibres emerge from each doublet and ensure anchoring of the edifice to the cell membrane ( Figure 1d). This gate function was formally demonstrated once molecular components of the TZ had been identified (Dean, Moreira-Leite, Varga, & Gull, 2016;Garcia-Gonzalo et al, 2011;Williams et al, 2011). Because of this arrangement, it has been proposed that the TZ constitutes a filter separating the flagellar compartment from the rest of the cell body (Gilula & Satir, 1972;Reiter, Blacque, & Leroux, 2012).…”

“…RNAi knockdown of several TZ components led to a loss of presence at the base of the new flagellum but not of the old one, showing that the two pools of TZ proteins are distinct and that there is no exchange between them(Dean et al, 2016). Furthermore, ablation of some TZ proteins impacted flagellum structure and composition to various extents(Dean et al, 2016;Dean, Moreira-Leite, & Gull, 2019;Harmer et al, 2018). It now remains to determine whether some TZ proteins show differential abundance, localisation, or function between growing and mature flagella.…”

“…Because of this arrangement, it has been proposed that the TZ constitutes a filter separating the flagellar compartment from the rest of the cell body (Gilula & Satir, 1972;Reiter, Blacque, & Leroux, 2012). This gate function was formally demonstrated once molecular components of the TZ had been identified (Dean, Moreira-Leite, Varga, & Gull, 2016;Garcia-Gonzalo et al, 2011;Williams et al, 2011). Mutations or loss of these molecules impact flagellum composition in multiple organisms including in humans where they are responsible for genetic diseases called ciliopathies (Reiter et al, 2012).…”