elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8

Abstract

The formation and function of cilia involves the movement of intraflagellar transport (IFT) particles underneath the ciliary membrane, along axonemal microtubules. Although this process has been studied extensively, its molecular basis remains incompletely understood. For example, it is unknown how the IFT particle interacts with transmembrane proteins. To study the IFT particle further, we examined elipsa, a locus characterized by mutations that cause particularly early ciliogenesis defects in zebrafish. We show here that elipsa encodes a coiled-coil polypeptide that localizes to cilia. Elipsa protein binds to Ift20, a component of IFT particles, and Elipsa homologue in Caenorhabditis elegans, DYF-11, translocates in sensory cilia, similarly to the IFT particle. This indicates that Elipsa is an IFT particle polypeptide. In the context of zebrafish embryogenesis, Elipsa interacts genetically with Rabaptin5, a well-studied regulator of endocytosis, which in turn interacts with Rab8, a small GTPase, known to localize to cilia. We show that Rabaptin5 binds to both Elipsa and Rab8, suggesting that these proteins provide a bridging mechanism between the IFT particle and protein complexes that assemble at the ciliary membrane.

Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin‐binding IFT‐B2 complex

“…Consistent with previously published data (Omori et al , 2008; Follit et al , 2009), we found that purified IFT54 associates with IFT20 via the coiled‐coil domain and not via the CH domain (Appendix Fig S2C and D). Similarly, the coiled‐coil regions of IFT38 and IFT57 were sufficient for IFT57/38 complex formation (Fig 2D, lane 5 and Appendix Fig S2E).…”

“…The N‐terminal CH domains of IFT57/38 mediate the contacts to IFT80 and IFT172, whereas the C‐terminal coiled‐coils allow for IFT57/38 hetero‐dimer formation and links to the IFT54/20 hetero‐dimer (Fig 2 and Appendix Fig S2). The interaction between IFT54 and IFT20 is in agreement with previously published data (Omori et al , 2008; Follit et al , 2009), whereas the interaction between IFT57 and IFT38 was not previously reported. Additionally, IFT57/38 mediates the contacts between the two IFT‐B subcomplexes by binding directly to IFT88/52N of IFT‐B1.…”

“…IFT38 localizes to the cilium and is required for ciliogenesis in a wide range of organisms, and mutations in IFT38 cause cystic kidneys in zebrafish and result in a loss of hedgehog signaling and embryonic lethality in mice (Sun et al , 2004; Stolc et al , 2005; Absalon et al , 2008). Furthermore, IFT38 was shown to undergo IFT in C. elegans (Ou et al , 2005) and co‐precipitated along with other IFT‐B proteins from zebrafish cell lysates using TAP‐IFT54 as a bait (Omori et al , 2008). To investigate how this protein associates with the IFT complex, we co‐expressed Cr IFT38 with different IFT‐B components resulting in soluble co‐expression only with the IFT57 subunit (Fig 1A and data not shown).…”

“…The IFT complex organizes into a 6 subunit IFT‐A and a 16 subunit IFT‐B subcomplex that separate in sucrose gradients even at low salt concentrations (Piperno & Mead, 1997; Cole et al , 1998; Lucker et al , 2005; Omori et al , 2008; Follit et al , 2009; Lechtreck et al , 2009b; Fan et al , 2010; Taschner et al , 2011; Ishikawa et al , 2014). Nine of the IFT‐B subunits (IFT88/81/74/70/52/46/27/25/22) were shown to assemble into a stable IFT‐B core (Lucker et al , 2005; Taschner et al , 2014).…”

Analysis of the retina in the zebrafish model

“…The heatshock promoter has been used, for example, to drive the expression of a crumbs gene from a Tol2-based vector in the zebrafish retinal neuroepithelium. This approach produced expression in nearly half of neuroepithelial cells (Omori et al, 2008).…”