A MOLECULAR ANALYSIS OF PROTEIN TRAFFICKING IN THE VERTEBRATE RETINA: IMPLICATIONS FOR INTRAFLAGELLAR TRANSPORT AND DISEASE

Vertebrate photoreceptors are highly specialized sensory neurons that utilize a modified cilium known as the outer segment to detect light. Proper trafficking of proteins to the outer segment is essential for photoreceptor function and survival and defects in this process lead to retinal disease. In this dissertation I focus on two aspects of protein trafficking, intracellular vesicular trafficking in photoreceptors and retinal pigmented epithelial (RPE) cells and how it relates to the human disease choroideremia (CHM), and the trafficking of proteins through the photoreceptor cilium. The human retinal degenerative disease choroideremia (CHM) is caused by mutation of the Rab escort protein-1 (REP1) gene, which is required for proper intracellular vesicular trafficking. However, it was unclear whether photoreceptor degeneration in this disease is cell-autonomous, due to defective opsin transport within the photoreceptor, or is noncell-autonomous and a secondary consequence of defective RPE. Utilizing the technique of blastomere transplantation and a zebrafish line with a mutation in the rep1 gene, I show that photoreceptor degeneration in CHM is noncell-autonomous and is caused by defective RPE. The molecular machinery responsible for protein trafficking through the photoreceptor cilium remained unclear for a long time. Recent studies found Intraflagellar Transport (IFT) is the process that mediates cilia formation and transport of proteins through a cilium, and further analyses showed IFT is important for trafficking proteins to the outer segment. However, many details about how IFT works in photoreceptors remained unclear. By analyzing zebrafish harboring a null mutation in the ift57 gene, I show that Ift57 is only required for efficient IFT, and that the Ift57 protein plays a role in the ATP-dependent dissociation of kinesin II from the IFT particle. Lastly, I investigate the role of retrograde IFT in photoreceptors, a process that had yet to be investigated. By utilizing antisense morpholino oligonucleotides to inhibit expression of cytoplasmic dynein-2 (the molecular motor that mediates retrograde IFT) , I show that retrograde IFT is required for outer segment extension and the recycling of IFT proteins

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

SUMMARY: 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

Dysfunction of Heterotrimeric Kinesin-2 in Rod Photoreceptor Cells and the Role of Opsin Mislocalization in Rapid Cell Death

Loss of kinesin-2 function causes rapid death of rod photoreceptors. The cell death is dependent on the expression of opsin, which first accumulates along the route to the outer segment, but not on signaling by opsin-arrestin complexes or by light activation; the key element appears to be the accumulation of excessive protein in the wrong place

The Par-PrkC polarity complex is required for cilia growth in zebrafish photoreceptors.

Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer's Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain

Pard3 expression is reduced at 5 dpf following injection of <i>pard3</i> morpholinos.

<p>(A–C) Transverse cryosections of wild-type retinas, (D–F) <i>pard3</i> morphant retinas and (G–I) control morphant retinas with Pard3 antiserum (left column) and the monoclonal antibody zpr-1 (middle column). The right panels show the merged images. Pard3 immunoreactivity was seen in the outer plexiform layer (OPL) and at cell junctions (A, arrowhead and arrows, respectively) in wild-type and control MO retinas but was significantly reduced in morphants retinas. All sections were also counterstained with DAPI (blue). ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer. Scale bar = 10 µm.</p

Left-right asymmetry requires <i>pard3</i>.

<p>(A) In situ hybridization for <i>southpaw</i> (<i>spaw</i>) expression in the lateral plate mesoderm in 18–20 somite stage embryos. <i>pard3</i> morphants show left-sided, right-sided, bilateral, or an absence of <i>spaw</i> expression. (B) Graphical summary from three independent experiments showing the percentage of control and morphant embryos with left-sided (L), right-sided (R), bilateral (B), or absence (A) <i>spaw</i> expression. The number (n) of embryos analyzed is given next to each set of bars. (C) KV cilia in a wild-type and <i>pard3</i> morphant. (D) Quantification of KV length across the longest axis. (E) Quantification of KV cilia number. (F) Quantification of KV cilia length. Bars show mean ± standard deviation (*p≤0.05; ***p≤0.0001) Scale bar = 20 µm.</p

Pharmacological inhibition of PrkC activity reduces cilia length and causes opsin mislocalization.

<p>(A–C) Transverse cryosections of 4 dpf retinas were stained with phalloidin to label actin (red) and 1D1 to label rhodopsin (green). (D–F) Immunohistochemistry on cryosections to label Ift88 in cilia (red) and acetylated tubulin to label ciliary microtubules (green). Arrows identify cilia. Sections were also counterstained with DAPI (blue). (G) Quantification of cilia length. Cilia length in 4 dpf wild-type photoreceptors (43.2 microns) and the non-myristoylated control inhibitor (48.9 µm) were not statistically different from each other, but both were statistically different from larvae treated with the PrkC inhibitor (27.1 microns). Error bars show standard deviation. (***p≤0.0001) Scale bar = 10 µm.</p

Connecting cilia are reduced in <i>pard3</i> morphants.

<p>(A–C) Transverse cryosections were stained for Ift88 (left, red) and acetylated tubulin (middle, green) to label cilia in 5 dpf retinas. Merged images are shown in right panels. Ift88 localizes to the connecting cilia emanating from the apical surface of photoreceptors (arrows). Staining is also seen in connecting cilia of UV cones (arrowheads), which are tiered below the other cones. Sections were also counterstained with DAPI (blue). ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer. Scale bar = 10 µm.</p

Photo-Morpholinos reduce apical domain size.

<p>(A–D) Transverse cryosections of 4 dpf retinas were stained with phalloidin to label actin (red) and zpr-1 to label double cones (green). Sections were also counterstained with DAPI (blue). (E) Quantification of apical domain as a percentage of total photoreceptor inner segment length. All values are plotted as a percentage of n/m, as defined in the text. Wild type = 38%; <i>pard3-pard3pMO</i> morphants = 16%; <i>prkci-prkcipMO</i> morphants = 9.72%; <i>prkc-prkcpMO</i> was non-detectable. Blue error bars indicate the mean ± standard deviation. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer. Scale bar = 20 µm.</p