Rab34 GTPase mediates ciliary membrane biogenesis in the intracellular ciliogenesis pathway
Preprint posted on October 29, 2020 https://www.biorxiv.org/content/10.1101/2020.10.29.360891v1
Article now published in Current Biology at http://dx.doi.org/10.1016/j.cub.2021.04.075
Primary cilia are microtubule-based organelles found at the cell surface where they perform key sensory and signalling functions. Their structure consists of a microtubule bundle, the axoneme, which extends from the mother centriole, and a ciliary membrane which is continuous with the plasma membrane. The ciliary membrane envelopes the axoneme and is bound to the distal appendages of the mother centriole at one end, which helps to maintain it as a distinct cellular compartment.
There are two pathways of cilium assembly. During extracellular ciliogenesis, the mother centriole docks at the plasma membrane prior to axoneme assembly, resulting in the growth of the cilium as a protrusion from the cell surface. On the other hand, during intracellular cilium assembly the mother centriole maintains its peri-Golgi localisation resulting in a cilium which is partly buried in the cell body with sometimes only the tip extending beyond the cell boundary.
Intracellular ciliogenesis is the better studied pathway (see diagram below). Here, small vesicles, likely derived from the Golgi and recycling endosomes, dock on to the distal appendages (distal appendage vesicles, DAVs) of the mother centriole. EHD family ATPases along with Pascin and SNAP29 then promote the remodelling and fusion of these DAVs to form the preciliary vesicle, which caps the end of the mother centriole. The axoneme then begins to assemble underneath this and as the microtubules grow, the membrane is expanded to wrap around the growing structure. This creates a double membrane layer along the microtubules with the inner layer, the ciliary membrane, contacting the axoneme and the outer layer, the ciliary sheath, facing the cytosol. Eventually the cilium reaches the cell surface where the ciliary sheath fuses with the plasma membrane creating an invagination at the cell surface, the ciliary pocket, in which the cilium sits.
Ciliary signalling is vital for development, tissue function and environmental sensing. It is thus not surprising that genetic mutations affecting cilium assembly or function have been linked to numerous debilitating disorders, collectively known as known as ciliopathies. As such, there is a strong clinical need to understand cilium biology.
In the presented study, Ganga et al investigate the role of a small GTPase, Rab34, in ciliogenesis. Interest in this protein originally stemmed from two CRISPR screens in which it was identified as a potential regulator of ciliary Hedgehog signalling1,2. Mutations in Rab34 have also been shown to cause ciliopathies in mice3,4. Here, the authors find that Rab34 knockout in NIH-3T3 and RPE1 cell reduces cilia numbers, further demonstrating a defect in ciliogenesis upon loss of Rab34 function.
There are a number of points at which ciliogenesis can stall or fail. To determine which step is affected by the loss of Rab34, the authors imaged knockout cells using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). 3D reconstructions of the mother centriole revealed the presence of multiple docked DAVs but no clear ciliary vesicle. A failure to recruit EHD1, Rab8 and ARL13B was also observed by light microscopy, however IFT proteins responsible for intraflagellar transport were still present and CP110, a centriolar capping protein removed prior to ciliogenesis, was absent. Altogether this indicates that ciliogenesis is initiated but stalls upstream of EHD1 activity due to a failure to fuse and remodel the membranes of the DAVs into a larger ciliary vesicle.
Using the GTP-locked and GTP-binding Rab34 mutants, Rab34-T66N and Rab34-Q111L, the authors next show that GTP hydrolysis is required for ciliogenesis. Furthermore, they identify two other residues in the Rab34 sequence that are conserved across species but not between Rab GTPases – S166 and L61. Mutation of these particular residues to Rab34-S166N and Rab34-L61D blocked the ability of Rab34 expression to rescue the knockout cell phenotypes. Furthermore, these mutants had a dominant negative effect in wildtype cells, not only causing DAVs to accumulate at the mother centriole, as seen in the knockouts, but also inducing DAV tubulation. This is consistent with a membrane remodelling function. Both Rab34-S166N and Rab34-L61D were identified as GTP-locked mutants trapped at the mother centriole.
Investigation of Rab34 localisation showed that Rab34 is selectively recruited to nascent cilia, although it can be present after an ARL13B-positive cilium has formed. Using antibody accessibility assays it was also found to exclusively localise to intracellular and not extracellular cilia. Consistent with a specific role in assembly of the former, knockout of Rab34 in MCDK cells, which only form extracellular cilia, did not affect ciliogenesis. One key feature of an intracellular cilium is the presence of the ciliary sheath. Using 3D structured illumination microscopy and expansion microscopy the authors establish that Rab34 localizes specifically to this membrane region. This is the first GTPase found to be enriched here.
I chose this paper because it provides clear evidence for distinct machinery that is differentially used by intra- and extracellular primary cilium assembly. Ciliopathies are highly diverse with respect to their pathologies and the affected tissues. Understanding the machinery involved in the formation of different types of cilia will help dissect the genetic mutations underlying these diseases and how the disease manifests. The observation that Rab34 specifically localises to the ciliary sheath also assigns this membrane domain a unique molecular identity, which has many implications for ciliary signalling and function.
- Breslow, D.K et al. 2018. A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nat Genet. 50:460-471.
- Pusapati, G.V. et al. 2018. CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog. Dev Cell. 44:113-129 e118.
- Xu, S. et al. 2018. Rab34 small GTPase is required for Hedgehog signaling and an early step of ciliary vesicle formation in mouse. J Cell Sci. 131:jcs213710
- Dickinson, M., Flenniken, A., Ji, X. et al. High-throughput discovery of novel developmental phenotypes. Nature 537, 508–514 (2016).
Posted on: 26th November 2020Read preprint
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