Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome

Abstract

Ciliopathies, including nephronophthisis (NPHP), Meckel syndrome (MKS) and Joubert syndrome (JBTS), can be caused by mutations affecting components of the transition zone, a ciliary domain near its base that controls the protein composition of its membrane. We defined the three-dimensional arrangement of key proteins in the transition zone using two-color stochastic optical reconstruction microscopy (STORM). NPHP and MKS complex components form nested rings comprised of nine-fold doublets. JBTS-associated mutations in RPGRIP1L or TCTN2 displace select transition zone proteins. Diverse ciliary proteins accumulate at the transition zone in wild type cells, suggesting that the transition zone is a waypoint for proteins entering and exiting the cilium. JBTS-associated mutations in RPGRIP1L disrupt SMO accumulation at the transition zone and the ciliary localization of SMO. We propose that the disruption of transition zone architecture in JBTS leads to a failure of SMO to accumulate at the transition zone, disrupting developmental signaling in JBTS.

Cell type‐specific regulation of ciliary transition zone assembly in vertebrates

“…In MEFs, mouse embryonic limb buds and mouse embryonic kidneys, Rpgrip1l deficiency perturbs the amount of members of the Nphp modules at the TZ (Figs A–E, and C–F and M–P), while the TZ amount of the analysed Mks/B9 proteins was unaltered in MEFs and mouse embryonic kidneys (Figs EV3A–E and G–K) bringing up the question whether Rpgrip1l is essential for TZ ultrastructure and TZ function as ciliary gatekeeper in these vertebrate cell types. Previously, it was shown that Rpgrip1l is arranged as discrete clusters in a ring‐like fashion within the TZ (Lambacher et al , ) and a recent report proposed that the components of the Mks/B9 and Nphp modules are localised at the arms of the Y‐links (Shi et al , ). We used TEM to analyse the ultrastructure of the TZ in Rpgrip1l −/− mouse embryonic kidneys and limb buds.…”

How the Ciliary Membrane Is Organized Inside-Out to Communicate Outside-In

“…Another type of superresolution microscopy, stochastic optical reconstruction microscopy (STORM), can achieve even higher resolution (~20 nm) than SIM. The high resolution capabilities of STORM enable the detection of spatially distinct proteins within subdomains of cilia, such as the relative positions of distinct ciliopathy-associated complexes within the transition zone [52]. The complementary use of cellular electron microscopy and super-resolution fluorescence microscopy will enable further advancement in our understanding of how subdomains of the ciliary membrane differ from each other.…”

“…In addition to functions as a diffusion barrier, recent super-resolution microscopy has revealed that the transition zone is a point at which many ciliary membrane-associated proteins accumulate, suggesting that this may be a waypoint at which proteins’ credentials are checked before being allowed entry or turned away (Figure 4) [52,78]. …”

“…Cells from Joubert syndrome individuals show defects in the architecture of the transition zone and, most probably consequently, defects in the protein composition of the ciliary membrane [52]. We have proposed that at least some forms of Joubert syndrome are due to defective transition zones causing problems in ciliary signaling.…”

“…A, The transition zone contains binding sites for select ciliary membrane proteins [52], and live imaging studies have shown that SMO pauses in the transition zone [78]. Thus, the transition zone may control the entry and exit of membrane-associated proteins from the cilium.…”