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. Author manuscript; available in PMC: 2013 Dec 15.
Published in final edited form as: Vision Res. 2012 Oct 23;75:37–43. doi: 10.1016/j.visres.2012.10.009

Analysis of KIF17 distal tip trafficking in zebrafish cone photoreceptors

Jason R Bader 1, Brandon W Kusik 1, Joseph C Besharse 1,*
PMCID: PMC3556995  NIHMSID: NIHMS423043  PMID: 23099049

Abstract

Multiple proteins are targeted to photoreceptor outer segments (OS) where they function in phototransduction. Intraflagellar transport (IFT), a highly conserved bidirectional transport pathway occurring along the microtubules of the ciliary axoneme has been implicated in OS trafficking. The canonical anterograde motor for IFT is the heterotrimeric kinesin II or KIF3 complex. Previous work from our laboratory has demonstrated a role for an additional kinesin 2 family motor, the homodimeric KIF17. To gain a better understanding of KIF17 function in photoreceptor OS we utilized transgenic zebrafish expressing zfKIF17-GFP to assess the localization and dynamics of zfKIF17. Our data indicate that both endogenous KIF17 and KIF17-GFP are associated with the axoneme of zebrafish cones at both early (5 dpf) and late (21 dfp) stages of development. Strikingly, KIF17-GFP accumulates at the OS distal tip in a phenomenon referred to as “tipping”. Tipping occurs in the large majority of photoreceptors and also occurs when mammalian KIF17-mCherry is expressed in ciliated epithelial cells in culture. In some cases KIF17-GFP is shed with the OS tip as part of the disc shedding process. We have also found that KIF17-GFP moves within the OS at rates consistent with those observed for IFT and other kinesins.

Keywords: Photoreceptors, Cilia, Kinesin, Intraflagellar transport, Zebrafish

1. Introduction

The outer segment (OS) of the vertebrate photoreceptor is a sensory cilium, which serves as a phototransduction organelle. Similar to other sensory cilia, the photoreceptor OS lacka a protein synthesis machinery, and their assembly and maintenance depends on intraflagellar transport (IFT). Originally identified in Chlamydomanas (Kozminski et al., 1993), this highly conserved process is characterized as a bi-directional trafficking pathway along a doublet microtubule (MT) backbone known as the ciliary axoneme. IFT has been proposed to deliver components involved in the light detection pathway to OS (Pazour et al., 2002; Bhowmick et al., 2009; Insinna et al., 2009). However, other mechanisms are involved and the mechanisms by which the cell regulates protein translocation to OS are not well understood. Because defects in this process have been linked to retinal degeneration and blindness, a better understanding of these mechanisms will provide critical insight into the photoreceptor degenerative diseases.

Several components are essential to the IFT process including microtubule-based motor proteins, and the multiprotein IFT complex, which is thought to function as a link between motor and cargo. IFT is driven by the kinesin (anterograde) and cytoplasmic dynein (retrograde) motors. Kinesins are motor proteins that use energy from ATP hydrolysis to drive transport of various cargoes along MTs. The heterotrimeric kinesin II complex (Marszalek et al., 2000), and more recently homodimeric KIF17 (Insinna et al., 2008; Insinna et al., 2009) have been identified as the motors responsible for anterograde IFT in photoreceptor OS.

KIF17 is a kinesin 2 family member that assembles as a homodimer (Signor et al., 1999; Setou et al., 2000) and has been implicated as a component of dendritic trafficking, spermatogenesis, epithelial morphogenesis, and ciliogenesis (reviewed in Wong-Riley and Besharse, 2012). KIF17 is the vertebrate homologue of OSM-3, one of two motors functioning in C. elegans chemosensory cilia (Signor et al., 1999). Disruption of OSM-3 leads to defects in chemosensory cilia function (Perkins et al., 1986; Starich et al., 1995), and recent work has suggested that OSM-3 is required for distal segment IFT and formation in chemosensory cilia (Snow et al., 2004; Evans et al., 2006; Hao, et al., 2011). Structurally, vertebrate photoreceptors are similar to C. elegans chemosensory cilia in that they have distal singlet MTs in their OS sensory cilium. Consistent with this, previous work from our laboratory demonstrated a unique role for KIF17 in OS formation (Insinna et al., 2008; Insinna et al., 2009).

Although we know that KIF17 plays an important role in OS formation, the spatial distribution of KIF17 in photoreceptor OS is not well understood. Localization of motor proteins and IFT components in photoreceptors has been limited to immunofluorescence studies. For the present study we generated transgenic zebrafish in which KIF17-GFP is expressed specifically in cone photoreceptors to assess the localization and dynamics of KIF17 in OS. Our data indicate that within the OS KIF17-GFP localizes to the axoneme and accumulates at the OS distal tip. In some cases distal KIF17-GFP is shed along with the distal tip and phagocytized by adjacent retinal pigment epithelial cells. Finally, using live cell imaging we have found that KIF17-GFP moves along the axoneme at rates consistent microtubule based motility and IFT.

2. Methods

2.1. Animals

Zebrafish were housed at 28.5°C on a 14-hr light:10-hr dark cycle. Embryos were raised at 28.5°C and placed in phenylthiourea to inhibit melanin synthesis during development and facilitate imaging. All experiments were approved and conducted in accordance with the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

2.2. Zebrafish transgenic lines

Cone specific expression of KIF17-GFP or Arl13b-mCherry was achieved by cloning the full length zebrafish KIF17 or mouse Arl13b cDNA fused at the C-terminus with GFP or mCherry downstream of the transducin alpha cone promoter (TaCP) (Kennedy et al., 2007; Insinna et al., 2009) using Gateway recombination (Invitrogen) with a Tol2kit (Kwan et al., 2007). Expression of the construct and the generation of transgenic lines were performed by injecting plasmid DNA and Transposase RNA (Kawakami, 2005) into one cell embryos with an injection volume of 4.6 nl/embryo. Embryos were screened for GFP expression and in some cases KIF17-GFP distribution was studied in embryos at 3–5 days after injection. Stable transgenic animals were obtained through outcrosses to WT animals to identify germline transmission of the transgene. Stable transgenic animals were advantageous because KIF-17 was expressed at similar levels in all cones.

2.3. Immunohistochemistry and microscopy

Cryosection analysis on 3–7 dpf zebrafish was performed as previously described (Insinna et al., 2008). When applicable, sections were immunostained with the mouse monoclonal zpr-1 antibody (1:200; ZFIN), mouse monoclonal zpr-2 antibody (1:200; ZFIN), or polyclonal antibodies against blue (BOPS) or green (GOPS) cone opsins (1:200; a gift from Dr. David Hyde, University of Notre Dame). Images were acquired on a Nikon C1 Plus-EX3 AOM Confocal System using the Nikon EZ-C1 software with a 60X 1.4-numerical aperture (NA) objective (Nikon Instruments, Melville, NY). Data processing was performed with ImageJ. Confocal images are displayed as maximum projections.

For analysis of live isolated photoreceptors, retinas were dissected from 5–21 dpf zebrafish and mechanically dissociated by gently pipetting in a zebrafish Ringer’s solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.2). Dissociated cells were plated in glass bottom dishes (MatTek Co., Ashland, MA) for live cell acquisition. Images were acquired on a microscope (Nikon Eclipse TE300; Nikon Instruments) operated with Metamorph software (MDS Analytical Technologies) and a camera (CoolSNAP HQ; Roper Scientific). Kymographs were generated in ImageJ with the kymograph plugin (J. Reitdorf). Fixation and staining of isolated photoreceptors was performed as previously described (Insinna et al., 2008). Isolated photoreceptors were immunostained with a mouse monoclonal antiacetylated-α-tubulin antibody (1:500, Sigma Aldrich, St. Louis, MO).

2.4. Mammalian cell culture

Pig kidney epithelial cells (LLC-PK1) were grown in DMEM/F12 (1:1) (Gibco) supplemented with 10% FBS and penicillin and streptomycin at 37°C in 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco). After transfection cells were serum starved for 24 hrs to induce ciliogenesis. Cells were fixed with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized with 0.2% Triton X-100 in PBS for 2 min and blocked in 3% BSA in PBS for 1 hr at RT. Cells were then incubated with primary antibodies in 3% BSA/PBS for 1hr at RT and after four washes with PBS were incubated with secondary antibodies in 3% BSA/PBS for 1 hr at RT. Following four washes with PBS, cells were mounted in Fluoromount-G (SouthernBiotech) for fluorescence microscopy.

3. Results

3.1 Generation and analysis of a KIF17-GFP line for localization and trafficking studies

Our previous work has localized endogenous KIF17 in both zebrafish and mouse photoreceptors to the inner segment with intense accumulation in the region of the basal body and has emphasized that within OS, KIF17 is associated with the ciliary axoneme (Insinna et al., 2008; Insinna et al., 2009). This work addresses the distribution of over expressed KIF17-GFP specifically in cone photoreceptors. The TaCP promoter for cone specific expression (Kennedy et al., 2007) was particularly valuable for our studies because it drives expression of transgenes in the majority of cones as early as 3–5 days postfertilization (dpf). At this stage of development, ~90% zebrafish retina photoreceptors are cones, which have already formed OS approaching adult proportions. Analysis of KIF17-GFP transgenic animals between 3 and 7 dpf revealed that KIF17-GFP expression is limited to the photoreceptor layer as seen by colabeling with zpr-1 (Fig. 1A). KIF17-GFP appears diffuse throughout the cell body, and the inner segment (IS), and is excluded from the nucleus (Fig. 1C). KIF17-GFP also appeared to localize prominently to the OS axoneme (Fig. 1C–D). To confirm this we used a second transgenic line, which expresses TaCP-Arl13b-mCherry, to label the OS. This useful marker labels cilia throughout development (Borovina et al., 2010). However, in zebrafish photoreceptors Arl13b-mCherry labels the OS diffusely. This is likely due to Arl13b being a membrane protein. In comparison to other types of cilia, the OS contains significantly more membranous material. KIF17-GFP signal overlaps with Arl13b-mCherry but is not distributed diffusely. It appears along axonemal structures and accumulates at the distal end of the OS (Fig. 1D). Co-labeling with blue cone opsin (Fig. 2D) and acetylated alpha tubulin reveals that KIF17-GFP’s OS distribution is associated with the axoneme in the OS (Fig. 2B). Although not pursued in this study, our early analysis of KIF17-GFP’s distribution in cone photoreceptors with transient transgenic larvae revealed that KIF17-GFP is expressed in photoreceptors of the pineal gland, some of which also express green cone opsin (Fig. 1B). This is consistent with previous work showing that in addition to retinal cones, TaCP drives expression in pineal photoreceptors (Kennedy et al., 2007).

Fig. 1.

Fig. 1

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Analysis of a transgenic zebrafish line with cone specific expression of KIF17-GFP. (A) Retinal cryosections of 3 dpf KIF17-GFP expressing zebrafish were stained with the zpr-1 antibody, an antibody that recognizes red-green double cones, to show the uniformity and photoreceptor specific expression of the TaCP driven transgene. Bar, 10 µm. (B) Pineal gland cryosection of 5 dpf transient transgenic KIF17-GFP expressing zebrafish was stained with the green cone opsin antibody (GOPS) and Hoechst. Bar, 20 µm. (C) Retinal cryosections of 5 dpf KIF17-GFP expressing zebrafish. KIF17-GFP signal is excluded from the nucleus (N) and is diffuse in the inner segment (IS). KIF17-GFP signal in the outer segment (OS) appears to concentrate on the axoneme (arrows). Bar, 10 µm. (D) Retinal cryosections of 5 dpf zebrafish co-expressing TaCP driven KIF17-GFP and Arl13b-mCherry. KIF17-GFP signal overlaps with Arl13b-mCherry confirming KIF17’s localization to the OS (arrows). Note that Arl13b-mCherry diffusely labels the OS. Bars, 10 µm.

Fig. 2.

Fig. 2

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KIF17-GFP translocates to the distal tip of cone photoreceptor OSs and is a component of disc shedding. (A) TaCP driven stable expression of KIF17-GFP in live, isolated zebrafish cones at 14 dpf. Transmitted light images reveal the structure of the OS. KIF17-GFP labels the OS axoneme and accumulates at the transition zone (arrow) and the photoreceptor OS distal tip (arrowhead). (B) Immunofluorescence microscopy analysis of KIF17-GFP in fixed isolated cone photoreceptors at 21 dpf. Photoreceptors were stained with an antibody against acetylated-α-tubulin (AcTub) to label the OS axoneme. KIF17-GFP accumulates at the basal body/transition zone (arrow) and the distal tip of the axoneme (arrowhead). (C) Immunofluorescence analysis of paraformaldehyde fixed LLC-PK1 cells expressing mCherry tagged KIF17 stained with an antibody to acetylated tubulin (green) to mark the position of the primary cilium and Hoechst (blue) to stain nuclei. KIF17-mCherry displays accumulation at the distal tip of the primary cilium (arrowheads). Note that the cilium is bent back on itself. (D) Retinal cryosections of KIF17-GFP expressing zebrafish prepared 5 days after injection of the KIF17-GFP construct. Note that these are not germline transgenic fish and KIF17-GFP is only expressed in a subset of the cones. (D; 1) Low power image of KIF17-GFP (green) co-stained with anti-blue cone opsin (red) and Hoechst for nuclei (blue). (D; 2–3) Higher power images of transient KIF17-GFP (green) eyes co-stained with ZPR2, which labels the RPE. Large arrows indicate cone OS axoneme labeling and arrowheads are possible phagosomes in the RPE. In 1 a phagosome (arrowhead) is labeled with both GFP and the blue cone opsin antibody while in 2–3 arrowheads indicate GFP containing bodies deep within RPE (red). All Bars, 10 µm.

3.2 Distal tip localization of KIF17-GFP in cone photoreceptors

Although 3–5 dpf zebrafish are useful for studying OS development, the size of the OS at these stages makes it difficult to fully assess protein localization. In order to overcome this, we looked at KIF17-GFP in our transgenic animals between 14 and 21 dpf, where OS are longer and easier to visualize. In addition, we took advantage of our ability to isolate photoreceptors from these animals to achieve better resolution in our imaging. Similar to 4–7 dpf animals, KIF17-GFP signal is diffuse within the cell body and IS. Interestingly, KIF17-GFP displays an accumulation at the IS-OS junction, presumably the transition zone or basal body, and the distal tip of cone photoreceptors (Fig. 2A). Isolated photoreceptors from our KIF17-GFP animals were fixed and labeled with an antibody against acetylated alpha tubulin to identify the axoneme (Fig. 2B). Co-labeling with acetylated tubulin reveals that KIF17-GFP accumulates at the distal tip of the OS axoneme (Fig. 2B) and we refer to this as “tipping”. Consistent with previous work (Dishinger et al., 2010), we found that mouse KIF17-mCherry could accumulate at the distal tip of primary cilia in kidney epithelial cells (Fig. 2C).

Because the tip of the OS is shed on a daily basis and OS derived phagosomes were evident in the RPE as early as 5 dpf, we next investigated whether KIF17-GFP is a component of this shed OS material. First, we co-labeled a subset of cone OS and phagosomes with an antibody to blue cone opsin (BOPS). This revealed that KIF17-GFP fluorescence was evident as phagocytosed material that co-labeled with BOPS in the RPE (Fig. 2D 1). We also identified KIF17-GFP labeled structures (phagosomes or OS tips) within RPE in which the cytoplasm was labeled with the antibody zpr-2 (Fig. 2D 2–3). This labeled the entire RPE cytoplasm. Despite the fact that the RPE contains many phagosomes at this early stage of development it was difficult to clearly distinguish KIF17-GFP labeled OS tips embedded in RPE from labeled phagosomes. In some cases, GFP labeled structures were clearly continuous with labeled axonemes (Fig. 2D-3; arrow and arrowhead). Thus, the frequency of shedding of tips containing KIF17-GFP remains uncertain and will require further analysis.

3.3 KIF17-GFP is a component of OS distal extensions OS

During our analysis of KIF17-GFP in 14–21 dpf isolated photoreceptor preparations we identified a unique structure in a subset of cones. This structure appears to be an extension of the OS axoneme that extends beyond the tip of the OS (Fig. 3A). Furthermore, it appears that in some instances the structure labeled with KIF17-GFP is contained within a structure visible in the bright field images that bulges off the side of the OS (Fig. 3B). To further investigate the organization of the axoneme itself we isolated photoreceptors expressing KIF17-GFP labeled them with an antibody against acetylated alpha tubulin (Fig. 3C–D). Staining with acetylated tubulin revealed that in some isolated cones there is a separation or splaying of the OS axoneme into two distinct structures within the OS (Fig. 3C). It is of interest that both the axoneme (originating in the IS) and the branching structure are labeled with acetylated alpha tubulin antibodies but that KIF17-GFP labeling and “tipping” is associated with the axoneme. These branching structures appear to be similar to accessory OS that have been reported in other teleost photoreceptors (Nagle et al., 1986).

Fig. 3.

Fig. 3

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Distal extensions and axonemal branching in zebrafish cone photoreceptors. (A–B) Fluorescent and brightfield images of live 21 dpf, isolated photoreceptors expressing KIF17-GFP. (A) KIF17-GFP labeling is apparent along a structure that extends distally beyond the photoreceptor tip (arrow) and accumulates at the tip of this structure (arrowhead). (B) A structure similar to that in A labeled with KIF17-GFP appears to diverge from the OS near the basal body or transition zone (arrow) and extends beyond the OS tip. KIF17-GFP does not display “tipping” in this structure. (C–D) Immunofluorescence analysis of KIF17-GFP expression in a cluster of 21 dpf cone photoreceptors. Staining with an antibody against acetylated-α-tubulin (AcTub) to visualize the OS axoneme, reveals a divergence or splaying of the axoneme (arrow). The boxed region in the brightfield image is blown up in D to more clearly show that axoneme divergence occurs in all 5 cones in this cluster of cells. This is most easily seen in the red image. KIF17-GFP distal tip accumulations (arrowhead) only occurs on one of the two axonemal structures (arrow) in a single OS (arrowhead). All bars, 5 µm.

3.4 Live-cell analysis of zfKIF17-GFP in isolated cone photoreceptors

Despite a few attempts direct visualization of MT-based transport in photoreceptors has remained elusive. To gain a better understanding of KIF17 dynamics in photoreceptor OS we utilized our transgenic animals to conduct live cell imaging of KIF17-based transport. With standard epifluorescent microscopy we have recently acquired evidence of KIF17-GFP particle motility in 5 dpf isolated cones from our transgenic lines (Fig. 4). In this assay KIF17-GFP labeled particles were observed moving toward the distal tip of the photoreceptor OS (Fig. 4); we did not detect retrograde movement. Kymograph analysis of particle tracks (N = 45 tracks in 30 cells) shows that KIF17-GFP particles move at rates (0.94 +/− 0.25 µm/sec) within a range expected for kinesin and IFT based motility. However, there was significant variability in rates measured at different positions along the axoneme and differences between proximal and distal domains of the axoneme were not detected. These data indicate that KIF17-GFP particles move in isolated photoreceptors at rates expected for microtubule-based transport.

Fig. 4.

Fig. 4

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Live cell analysis of KIF17-GFP particle motility in zebrafish cones. (A;1–8) 14 sec time-lapse series of KIF17-GFP particle motility (4–18 sec, lower left) in isolated 5 dpf cone OS. Arrow denotes small GFP tagged particle. N, nucleus; IS, inner segment; OS, outer segment. Bar, 10 µm. (B) Enlargement of 1–8 to make particle more apparent. Position of arrow tracks small motility changes in a GFP tagged particle. (C) Kymographic analysis of KIF17-GFP particle motility with a corresponding diagram depicting particle trajectories (diagonal tracks). Only anterograde tracks were detected in this abalysis. The verticle bar on the kymograph image corresponds to 14 sec and the horizontal bar to 2.5 µm.

4. Discussion

Previous work from our laboratory identified a role for KIF17 in vertebrate photoreceptor OS formation (Insinna et al., 2008; Insinna et al., 2009). However, the direct contribution of KIF17 to photoreceptor ciliogenesis and OS formation is not clear. In order to gain a better understanding of KIF17 function in vertebrate photoreceptor OSs and KIF17’s predicted role in IFT, it is necessary to fully assess the spatial characteristics of the motor. The data presented here provide important insight regarding the spatial distribution of KIF17 in vertebrate photoreceptor OS. One intriguing aspect of our findings is the tipping behavior of KIF17-GFP. This localization pattern was not surprising based on previous work suggesting that KIF17 can accumulate at the distal tip of primary cilia in cultured mammalian cell lines (Dishinger et al., 2010). Our work with mammalian cells and photoreceptors supports this and suggests a conserved mechanism for distal ciliary targeting of KIF17.

Our demonstration that KIF17-GFP accumulates at the tip of the photoreceptor OS/axoneme is important in that it also supports a potential role in distal MT singlet assembly in vertebrate photoreceptors. This is consistent with the finding that OSM-3 is required for distal MT singlet assembly in C. elegans chemosensory cilia (Perkins et al., 1986; Snow et al., 2004; Evans et al., 2006; Hao, et al., 2011). Furthermore, a predicted loss-of-function kif17 mutation in zebrafish has recently been shown to induce a truncation of olfactory cilia (Zhao et al., 2012). Although shortened, these olfactory cilia still have distal MT singlets (Zhao et al., 2012). However, it is not clear whether KIF17 is responsible for the formation or elongation of distal singlets in this subset of cilia.

Aside from a proposed role in distal singlet formation/extension, one might speculate that KIF17 may play a role in OS disc shedding. The tip of the OS is shed as a part of the overall OS turnover process and the shed material is phagocytosed by the adjacent RPE (Young and Bok, 1969). The mechanisms controlling this system are not well understood. Our data suggests that KIF17-GFP is a component of shed material phagocytosed by the RPE. These findings along with KIF17’s functions in singlet elongation and plus end stability suggest a role in the photoreceptor disc shedding pathway. However, this notion requires further investigation.

The translocation of KIF17 to the distal tip of the vertebrate photoreceptor axoneme may provide clues in regards to specific cargo being transported to the tip of the photoreceptor. KIF17 has been shown to target CNG (cyclic nucleotide-gated) channels to the primary cilium in both kidney epithelial cells and olfactory sensory neurons (Jenkins et al., 2006). CNG channels have been shown to accumulate in the distal segment of frog olfactory cilia (Flannery et al., 2006). Numerous studies have characterized the importance of CNG channels to photoreceptor function and the idea of a photoreceptor CNG channel being a KIF17-specific cargo requires further elucidation.

Another interesting aspect of this work is the finding that zebrafish cone photoreceptors appear to extend their axonemes beyond the tip of the OS. Although we cannot rule out the possibility that these structures are artifacts resulting from the dissociation process, KIF17-GFP is clearly of component of these structures, and is capable of “tipping” in these structures. In addition, we identified a structure that appears to diverge from the OS in the proximal region of the OS that may correspond to the previously reported accessory OS (Nagle et al., 1986). The observation that KIF17-GFP is only observed in association with one of these structures in a single OS suggests that they serve different functions within the OS. The function of these branching structures is unclear, but accessory OSs have been proposed to provide structure for retinomotor movements and/or mediate metabolic exchange between photoreceptors and the RPE (Engström, 1963). These structures were not observed on all isolated photoreceptors and it will be interesting to investigate their function and whether they are unique to a specific subset of cones. It is possible, however, that such structures are damaged or lost in many cells during our isolation procedure.

Our early attempts at directly visualizing KIF17-GFP transport in photoreceptors appear promising, and should provide us with a plethora of information in regards to the dynamics of MT-based transport and IFT in photoreceptors. Velocity measurements indicate that KIF17-GFP moves at a rate consistent with microtubule-based transport (0.94 +/− 0.25 µm/sec) along axoneme. However, it is not clear from these studies whether KIF17-GFP is the motor responsible for the movement as opposed to cargo. It is of interest to note that velocity measurements exhibited significant variability, and we did not detect significant differences in velocity between proximal and distal regions of the OS axoneme. Future studies should be aimed at utilizing imaging techniques including but not limited to fluorescence recovery after photobleaching (FRAP), photoactivation, and fluorescence resonance energy transfer (FRET). FRAP analysis will be especially useful in visualizing KIF17 distal tip trafficking. Although zebrafish are an ideal live imaging model recent work has made significant headway in achieving live cell analysis of IFT in mouse photoreceptors (Trivedi et al., 2012).

Although recent work with a predicted loss-of-function kif17 mutation in zebrafish suggests a minimal role for KIF17 in vertebrate OS formation (Zhao et al., 2012), it is unclear whether this mutation induces a true null. The data presented here coincides with our previous work (Insinna et al., 2008) and supports the idea that KIF17 plays an important role in OS trafficking and formation. An analysis to be presented separately (Bader, et al., in preparation) suggests that the kif17 mutation reported on previously (Zhao, et al., 2012) is not a null allele and that when KIF17 is knocked down completely, OS fail to form during early development.

Supplementary Material

01

Video 1. KIF17-GFP particle motility in zebrafish cones. Time-lapse image sequence was collected at 1 frame per second and played in video at 7 frames per second. Stills from the time-lapse sequence are presented in Fig. 4. Bar, 5 µm.

Download video file (131.8KB, mov)

Highlights of this Paper.

We have developed a line of zebrafish expressing KIF17-GFP specifically in cones.

Over-expressed KIF17-GFP associates with the axoneme and accumulates at its tip.

“Tipped” KIF17-GFP can be shed with the OS tip and phagocytized by RPE.

KIF17-GFP is found in novel distal and lateral extensions of cone axonemes.

In isolated cones KIF17-GFP moves distally at rates expected for kinesin motors.

Acknowledgements

This work was supported by National Institutes of Health (NEI) Grant EY03222 (JCB), a grant from Advancing a Healthier Wisconsin at the Medical College of Wisconsin and NEI Core grant P30EY01931.

Footnotes

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Associated Data

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Supplementary Materials

01

Video 1. KIF17-GFP particle motility in zebrafish cones. Time-lapse image sequence was collected at 1 frame per second and played in video at 7 frames per second. Stills from the time-lapse sequence are presented in Fig. 4. Bar, 5 µm.

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