The intraflagellar transport protein IFT57 is required for cilia maintenance and regulates IFT-particle–kinesin-II dissociation in vertebrate photoreceptors

Abstract

Defects in protein transport within vertebrate photoreceptors can result in photoreceptor degeneration. In developing and mature photoreceptors, proteins targeted to the outer segment are transported through the connecting cilium via the process of intraflagellar transport (IFT). In studies of vertebrate IFT, mutations in any component of the IFT particle typically abolish ciliogenesis, suggesting that IFT proteins are equally required for IFT. To determine whether photoreceptor outer segment formation depends equally on individual IFT proteins, we compared the retinal phenotypes of IFT57 and IFT88 mutant zebrafish. IFT88 mutants failed to form outer segments, whereas IFT57 mutants formed short outer segments with reduced amounts of opsin. Our phenotypic analysis revealed that IFT57 is not essential for IFT, but is required for efficient IFT. In co-immunoprecipitation experiments from whole-animal extracts, we determined that kinesin II remained associated with the IFT particle in the absence of IFT57, but IFT20 did not. Additionally, kinesin II did not exhibit ATP-dependent dissociation from the IFT particle in IFT57 mutants. We conclude that IFT20 requires IFT57 to associate with the IFT particle and that IFT57 and/or IFT20 mediate kinesin II dissociation.

Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

“…Interestingly, although maternal ift57 transcripts decay by 33 hpf in mutants ( Figure 1D), previous studies indicate that Ift57 protein can be detected at 48 hpf, but not at 4 dpf, in ift57 hi3417 mutants. 26,34 Together, these results suggest that maternally supplied Ift57 protein or transcript can persist for an extensive period of time. Consistent with the gradual degradation of maternal gene products, cilia assembly is initially present on the first day of development in ift57 hi3417 and ift172 hi2211 mutants, but is lost at later stages of development.…”

“…Proteins were transferred to nitrocellulose membranes. Filters were then blotted in PBS buffer with 5% instant milk, incubated sequentially with anti-Ift57 (kindly provided by B. Perkins) 34 at 1:2500 and horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) at 1:5000 to 1:10,000. Signals were then detected with enhanced chemiluminescence detection using the Western Lightning kit from PerkinElmer Life Sciences.…”

Biology of Cilia and Ciliopathies

“…The current model suggests IFT20 is involved in directing vesicles transporting ciliary-specific proteins near the basal body, and participating in the trafficking of membrane proteins into the flagellar compartment [40]. IFT57 can target to the transition fibers of the axoneme, and serves as an anchoring protein for IFT20 to IFT-B in zebrafish [41]. IFT172 is also an interesting protein, since it readily dissociates from the IFT particle and has been shown to be important for retrograde movement.…”

Analysis of the retina in the zebrafish model

“…Simple test of vision based on swimming behavior Easter and Nicola (1996) Electrophysiological tests ERG Test of retinal function based on the detection of electrical activity of retinal neurons and glia Avanesov et al (2005), Brockerhoff et al (1995) Biochemical approaches Co-immunoprecipitation from embryo extracts Identification of direct and indirect protein binding partners Insinna et al (2008), Krock and Perkins (2008) Tandem affinity purification from embryo extracts…”