The nphp-2 and arl-13 Genetic Modules Interact to Regulate Ciliogenesis and Ciliary Microtubule Patterning in C. elegans

Abstract

Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—nphp-2+klp-11 and arl-13+unc-119—which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

“…One candidate is the nuclear import machinery, as there are lines of evidence suggesting that components of the nuclear import machinery, including the Ran GTPase and importins, are involved in the entry of proteins through the transition zone into cilia (Dishinger et al, 2010;Hurd et al, 2011). It will also be interesting to investigate whether transition zone proteins, which when mutated result in ciliopathies including Joubert syndrome (Garcia-Gonzalo et al, 2011;Warburton-Pitt et al, 2014), are directly involved in the ciliary entry of ARL13B. Recently, Slaats et al reported that ARL13B and INPP5E are markedly reduced in the cilia of fibroblasts derived from JBTS patients in which the MKS1 gene is mutated (Slaats et al, 2016).…”

Structural basis of the Inv compartment and ciliary abnormalities inInv/nphp2mutant mice

“…Therefore, the length of the microtubule doublet region is similar to that of the Inv compartment plus the transition zone. In Caenorhabditis elegans , the inv homolog is localized in the middle portion of the cilia, which consists of the microtubule doublet region [Perkins et al, ; Warburton‐Pitt et al, ]. Thus, the Inv compartment of renal cilia is quite likely to correspond to the microtubule doublet region of C.elegans .…”

Arl13b regulates Shh signaling from both inside and outside the cilium

“…Intriguingly, this duplication coincides with the vertebrate-specific link between cilia and Hedgehog signaling (Logsdon and Kahn, 2003; Li et al ., 2004; Kahn et al ., 2008; East et al ., 2012; Schlacht et al ., 2013). In Caenorhabditis elegans , which lacks, Hedgehog signaling, arl-13 mutations disrupt cilia structure and ciliary protein traffic (Cevik et al ., 2010; Li et al ., 2010; Warburton-Pitt et al ., 2014). Although no functional or loss-of-function mutations have been reported for Arl13a orthologues, mutations in Arl13b cause a number of defects in humans and model organisms, such as developmental defects in zebrafish (Sun et al ., 2004; Duldulao et al ., 2009).…”