The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development

Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.     

Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.     

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.     

Immunologic characterization of the photoreceptor outer segment cyclic GMP phosphodiesterase

High affinity (KD approximately 1 X 10(-9) M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed greater than 95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retina extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.     

Serine phosphorylation regulates paxillin turnover during cell migration

Background

Paxillin acts as an adaptor protein that localizes to focal adhesion. This protein is regulated during cell migration by phosphorylation on tyrosine, serine and threonine residues. Most of these phosphorylations have been implicated in the regulation of different steps of cell migration. The two major phosphorylation sites of paxillin in response to adhesion to an extracellular matrix are serines 188 and 190. However, the function of this phosphorylation event remains unknown. The purpose of this work was to determine the role of paxillin phosphorylation on residues S188 and S190 in the regulation of cell migration.

Results

We used NBT-II epithelial cells that can be induced to migrate when plated on collagen. To examine the role of paxillin serines 188/190 in cell migration, we constructed an EGFP-tagged paxillin mutant in which S188/S190 were mutated into unphosphorylatable alanine residues. We provide evidence that paxillin is regulated by proteasomal degradation following polyubiquitylation of the protein. During active cell migration on collagen, paxillin is protected from proteasome-dependent degradation. We demonstrate that phosphorylation of serines 188/190 is necessary for the protective effect of collagen. In an effort to understand the physiological relevance of paxillin protection from degradation, we show that cells expressing the paxillin S188/190A interfering mutant spread less, have reduced protrusive activity but migrate more actively.

Conclusion

Our data demonstrate for the first time that serine-regulated degradation of paxillin plays a key role in the modulation of membrane dynamics and consequently, in the control of cell motility.     

IFT20 is required for opsin trafficking and photoreceptor outer segment development

The light-detecting outer segments of vertebrate photoreceptors are cilia. Like other cilia, all materials needed for assembly and maintenance are synthesized in the cell body and transported into the cilium. The highly elaborated nature of the outer segment and its high rate of turnover necessitate unusually high levels of transport into the cilium. In this work, we examine the role of the IFT20 subunit of the intraflagellar transport (IFT) particle in photoreceptor cells. IFT20 was deleted in developing cones by a cone-specific Cre and in mature rods and cones by a tamoxifen-activatable Cre. Loss of IFT20 during cone development leads to opsin accumulation in the inner segment even when the connecting cilium and outer segment are still intact. With time this causes cone cell degeneration. Similarly, deletion of IFT20 in mature rods causes rapid accumulation of rhodopsin in the cell body, where it is concentrated at the Golgi complex. We further show that IFT20, acting both as part of the IFT particle and independent of the particle, binds to rhodopsin and RG-opsin. Since IFT20 dynamically moves between the Golgi complex and the connecting cilium, the current work suggests that rhodopsin and opsins are cargo for IFT transport.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named “phototransductive plasma membrane domain," and the latter to be named “disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes. For proper localization of cGMP-gated channel to the phototransductive plasma membrane, the glutamic acid-rich protein domain encoded in the β subunit plays a critical role. Quantitative study suggests that the disk morphogenic domain likely plays an important role in enriching rhodopsin prior to its sequestration into closed disk membranes. Thus, this and our previous studies provide new insight into the mechanism that spatially organizes the vertebrate phototransduction cascade.     

Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development

N-cadherin, a member of the cadherin family of calcium-dependent cell adhesion molecules, mediates adhesive and signaling interactions between cells during development. N-Cadherin undergoes dynamic spatiotemporal changes in expression which correlate with morphogenetic movements of cells during organogenesis and histogenesis. We have previously shown that N-cadherin expression during development is regulated by several mechanisms, including mRNA expression, cytokine modulation, and proteolytically mediated turnover, yielding the NCAD90 protein. The present study was directed at determining the extent to which N-cadherin in primary embryonic cells is the target of endogenous kinases and phosphatases, as well as the effects of modulation of these enzymes on NCAD90 expression. The results of phosphoamino acid analyses, peptide mapping, and measurements of N-cadherin and NCAD90 expression in embryonic tissues indicate that N-cadherin is indeed the target of endogenous kinase and phosphatase action, and that modulation of different classes of these enzymes can result in either stimulation or inhibition of NCAD90 production. These results provide a mechanistic explanation for observations that cadherin function is downregulated following expression of exogenously introduced viral tyrosine kinases and provide a function for the tyrosine phosphatases recently found in association with cadherins. The results indicate that N-cadherin expression during retinal development is possibly regulated in part by modulation of its phosphorylation state, the balance of which may determine whether N-cadherin remains stably expressed or is targeted for proteolytically mediated turnover to produce NCAD90. Dev. Genet. 20:224–234, 1997. © 1997 Wiley-Liss, Inc.     

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.     

Light-dependent association of Src with photoreceptor rod outer segment membrane proteins in vivo.

In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.     

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.     

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

In a previous study, we used differential proteomics to identify retinal proteins whose steady‐state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady‐state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina—retinal recycling and generation of retinoic acid—we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11‐cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 623–635, 2010     

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Functional expression of oxidative phosphorylation proteins in the rod outer segment disc

The rod Outer Segment (OS) disc, an organelle devoid of mitochondria, is specialized in phototransduction, a process requiring a continual chemical energy supply. We have shown that OS discs express functional mitochondrial electron transport chains, F_oF₁‐ATP synthase and the tricarboxylic acid cycle enzymes, all mitochondrial features. Here, we focus on oxygen consumption and adenosine triphosphate (ATP) synthesis by OS discs analysing electron transport chain I‐III‐IV and II‐II‐IV pathways, supported by reduced nicotinamide adenine dinucleotide and succinate, respectively. Interestingly, respiratory capacity of discs was measurable also in the presence of 3‐hydroxy‐butyrrate, a typical metabolic substrate for the brain. Data were supported by a two‐dimensional electrophoresis analyses conducted as our previous one, but focused to those mitochondrial proteins that are involved in oxidative phosphorylation. Carbonic anhydrase was also found active in OS discs. Moreover, colocalization of Rhodopsin with respiratory complex I and ATP synthase seems a further step in the characterization of some proteins typical of the mitochondrial inner membranes that are expressed in the rod discs. The existence of oxygen utilization in the outer retina, likely supplying ATP for phototransduction, may shed light on some retinal pathologies related to oxidative stress of the outer retina. Copyright © 2013 John Wiley & Sons, Ltd.     

Photobleaching and cyclic GMP dependences of rhodopsin phosphorylation in rod outer segment

The experimental data on the cGMP decrease under continuous illumination of rod outer segment have been theoretically analysed to study the bleaching and hence the cGMP dependence of the rhodopsin phosphorylation. From the agreement of the theoretical results with the experimental observations it has been found that the rate of phosphorylation depends on the rate of cGMP hydrolysis. If the rate of cGMP hydrolysis increases the rate of phosphorylation also increases. The results of the theoretical treatment predict that (i) the presence of cGMP in rod outer segment inhibits the rhodopsin phosphorylation and (ii) rhodopsin phosphorylation process is much faster than what has been reported in the literature.     

Proteolytic shedding of the extracellular domain of photoreceptor cadherin. Implications for outer segment assembly

Photoreceptor cadherin (prCAD) is a distinctive cadherin family member that is concentrated at the base of rod and cone outer segments and is required for their structural integrity. During retinal development, prCAD localizes to the site of the future outer segment before rhodopsin or other phototransduction proteins. In vivo, prCAD undergoes a single proteolytic cleavage that releases the ectodomain as a soluble fragment. The C-terminal fragment containing the transmembrane and cytosolic domains remains associated with the outer segment. In rds(-/-) retinas, in which outer segment assembly is severely disrupted because of the absence of retinal degeneration slow (RDS)/peripherin, an essential outer segment structural protein, the level of prCAD is increased, whereas the levels of other outer segment proteins are decreased relative to wild type retinas. Additionally, the ratio of intact:cleaved prCAD polypeptides is increased in rds(-/-) retinas. These data imply that prCAD ectodomain cleavage is an integral part of the outer segment assembly process, and they further suggest that outer segment assembly might be driven, at least in part, by the near irreversibility of proteolysis.     

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.