An Assay for Clogging the Ciliary Pore Complex Distinguishes Mechanisms of Cytosolic and Membrane Protein Entry

Abstract

Summary As a cellular organelle, the cilium contains a unique protein composition [1, 2]. Entry of both membrane [3–5] and cytosolic components [6–8] is tightly regulated by gating mechanisms at the cilium base, however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs) which comprise a ciliary pore complex (CPC) [6, 9]. To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC) [10], for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated active transport of bovine serum albumin into the nuclear compartment and of the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle’s limiting membrane.

Endocytic Control of Cellular Signaling at the Primary Cilium

“…However, this result was questioned in a recent study where ciliary SMO localization was found to be unaffected by simultaneous loss of b-arrestins 1 and 2 [53]. Further, single-molecule studies have indicated that SMO is translocated laterally from the plasma membrane into the ciliary compartment and, once inside the cilium, it primarily moves by diffusion and not IFT [22][23][24][25]. Similarly, two separate studies in Chlamydomonas have shown that specific membrane proteins can accumulate in the cilia of that organism independently of IFT [26,54].…”

“…Studies in multiple organisms have shown that ciliary assembly and maintenance rely on the vesicular transport of lipids and proteins from the trans-Golgi network (TGN) and recycling endosomes to the mother centriole/basal body, where vesicles are exocytosed and lipids and proteins are incorporated into the growing ciliary membrane [21]. Some ciliary membrane proteins, such as SMO of the Shh signaling pathway, may also enter the ciliary compartment by lateral transport or diffusion from the plasma membrane [22][23][24][25] ( Figure 1A). The pathways controlling vesicle and membrane protein trafficking to the cilium are complex and not fully understood.…”

The essential roles of transition fibers in the context of cilia

“…After docking, the proposed ciliary pore complex (CPC), possibly positioned at inter-fiber space of TFs, facilitates the import of assembled IFT particles. The molecular identities of the CPC remain mysterious, and molecular evidence supports [40, 51, 52] or challenges [42] the similarity of the CPC and NPC. It is worth determining if this active transport mechanism applies to other non-IFT ciliary proteins, how the CPC recognizes the cilia-targeting signal of ciliary proteins, and what is the functional and structural relationship among the CPC, TFs, and the TZ.…”

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

“…Many GFP-tagged nucleoporins localize to the base of the cilium, and introduction of inhibitors of NPC diffusion perturb the size-exclusion permeability barrier at the base of the cilium (Kee et al, 2012). Moreover, forced dimerization of Nup62 perturbs the directional trafficking of cytosolic proteins into the cilium, but has no effect on membrane proteins (Takao et al, 2014). Studies by other groups have shown functional roles for the nucleoporins Nup62 and Nup188 at the centrosome, which contributes its mother centriole to form the ciliary basal body (Hashizume et al, 2013;Itoh et al, 2013).…”

“…It has been proposed that nucleoporins localize to the base of the cilium to form a complex that fulfills a role similar to that of the NPC, possessing activity as both a diffusion barrier and a regulator of directional import into the cilium (Kee et al, 2012;Takao et al, 2014). Many GFP-tagged nucleoporins localize to the base of the cilium, and introduction of inhibitors of NPC diffusion perturb the size-exclusion permeability barrier at the base of the cilium (Kee et al, 2012).…”