Figure 4 - uploaded by Wolfgang Baehr
Content may be subject to copyright.
Fine structure of Kif3a-deleted cones at postnatal day 13. Proximal COSs reveal that connecting cilia (black arrows; a– c) develop, although outer segment membranes appear misaligned. Misalignment was most severe in cones inferior to the optic nerve, and less severe in cones located in the superior midperiphery. Examples of COSs found in various stages of membrane disorganization: a, immediately inferior to the optic nerve and advanced; b, c, intermediate; and d, e, early in degeneration. The plasma membrane (dashed white line; d, f ) defines the extent of the distal cone inner segment (CIS), in which cone mitochondria (m c ) with disrupted cristae were distinct from rod mitochondria (m r ). A cascade of five to seven overlapping membrane discs appears at the expanded axoneme (between pair of white arrows; d, e). More proximally, in a c Kif3a / cone inner segment (f, between dashed white lines), vesicles of heterogeneous diameter were dispersed among mitochondria (m c ) and a Golgi apparatus. Scale bar, 0.2 m (for a–f ).
Source publication
Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking K...
Contexts in source publication
Context 1
... determine the effect of KIF3A deletion on COS fine structure, 60-nm-thin sec- tions of P13 c Kif3a / retina, taken in the central retina inferior to the optic nerve, were analyzed by electron microscopy (Fig. 4). The P13 time point, 1 d after eye opening, was chosen to capture structural deficiencies before massive degeneration. Cones were identified by large oval nuclei containing a clump of finely granular heterochromatin and, oc- casionally, an osmiophilic droplet in the inner segment (supple- mental Fig. S4, white arrow, available at ...
Context 2
... were analyzed by electron microscopy (Fig. 4). The P13 time point, 1 d after eye opening, was chosen to capture structural deficiencies before massive degeneration. Cones were identified by large oval nuclei containing a clump of finely granular heterochromatin and, oc- casionally, an osmiophilic droplet in the inner segment (supple- mental Fig. S4, white arrow, available at www.jneurosci.org as supplemental material); rod nuclei, in contrast, were much more electron dense. Cone outer segments were observed in various stages of degeneration with membrane layers found in both per- pendicular and parallel orientations as well as in whorls (Fig. 4a-c). Connecting cilia of affected ...
Context 3
... droplet in the inner segment (supple- mental Fig. S4, white arrow, available at www.jneurosci.org as supplemental material); rod nuclei, in contrast, were much more electron dense. Cone outer segments were observed in various stages of degeneration with membrane layers found in both per- pendicular and parallel orientations as well as in whorls (Fig. 4a-c). Connecting cilia of affected cones appeared to have nor- mal structures proximally, suggesting that KIF3A is not required for ciliary maintenance at P13. However, overlapping disc cas- a, c, e, g, i, k) and c Kif3a / retinas (b, d, f, h, j, l ) were probed with antibodies recognizing CNGA3 (a, b), GC1 (c, d), GCAP1 (e, f ), prominin-1 ...
Context 4
... (green). The ONL of some sections was contrasted with propidium iodide (a-f, i-l ), whereas in other sections, cone cells were identified with anti-cone arrestin (red; g, h). Note that, in c Kif3a / cones, membrane proteins were severely reduced or completely absent, with the exception of peripherin/rds (l, arrows). Scale bar, 10 m. cades (Fig. 4d,e, between white arrows) and longitudinally aligned membranes were sometimes observed at the distal end of connecting cilia of early degenerating cones. c Kif3a / cone inner segments contained distended Golgi apparati and mitochondria typically characterized by a pale matrix with irregularly arranged cris- tae (Fig. 4d,f, m c ). Whereas ...
Context 5
... Scale bar, 10 m. cades (Fig. 4d,e, between white arrows) and longitudinally aligned membranes were sometimes observed at the distal end of connecting cilia of early degenerating cones. c Kif3a / cone inner segments contained distended Golgi apparati and mitochondria typically characterized by a pale matrix with irregularly arranged cris- tae (Fig. 4d,f, m c ). Whereas inner and outer segments showed obvious signs of dysfunction, cone pedicles remained vesicle-laden with multiple, small synap- tic contacts (supplemental Fig. S5, white arrows, available at www.jneurosci.org as supplemental ...
Context 6
... sembled and transported through the ci- lium by an anterograde molecular motor. Our results indicate that the anterograde molecular motor primarily responsible for IFT in cone photoreceptors is hetero- trimeric kinesin-II. Deletion of the sub- unit KIF3A motor results in the following defects: Misorganization of the outer segment membrane at P13 (Fig. 4), mislo- calization of visual pigments ( Fig. 1; supple- mental Fig. S1, available at www.jneurosci. org as supplemental material), and absence of membrane-associated polypeptides of the cone phototransduction cascade (Fig. 2), as well as integral outer segment mem- brane proteins (Fig. 3). In c Kif3a / cones, S-and M/L-opsins appear ...
Similar publications
Prominin-1 expresses in rod and cone photoreceptors. Mutations in the prominin-1 gene cause retinal degeneration in humans. In this study, the authors investigated the expression and subcellular localization of xlProminin-1 protein, the Xenopus laevis ortholog of prominin-1, in rod and cone photoreceptors of this frog.
Antibodies specific for xlPro...
Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The mole...
Citations
... Ultrastructural defects in rod OSs were not detected; these may be rare due to the presence of functional GUCY2F in rods. A similar ultrastructural phenotype of misaligned OS disks combined with mislocalized OS proteins and progressive photoreceptor cell loss has been observed in several mouse retinal dystrophy models bearing mutations in genes involved in OS development and maintenance, including Cngb1-Garp1-Garp2 (Zhang et al., 2009), Kif3a (Avasthi et al., 2009), Prom1 (Zacchigna et al., 2009;Dellett et al., 2014), and Rp1 (Gao et al., 2002;Liu et al., 2003;Song et al., 2014). As recently summarized (Goldberg et al., 2016), the proteins encoded by these genes are implicated in cargo transport across the connecting cilium (KIF3A), disk formation (CNGB1-GARP1-GARP2, PROM1, RP1), disk stacking (RP1), and disk stabilization (CNGB1-GARP1-GARP2). ...
During routine screening of mouse strains and stocks by the Eye Mutant Resource at The Jackson Laboratory for genetic mouse models of human ocular disorders, we identified cpfl9, a mouse model with cone photoreceptor function loss. The mice exhibited an early-onset phenotype that was easily recognized by the absence of a cone-mediated b-wave electroretinography response and by a reduction in rod-mediated photoresponses at four weeks of age. By genetic mapping and high-throughput sequencing of a whole exome capture library of cpfl9, a homozygous 25 bp deletion within exon 11 of the Gucy2e gene was identified, which is predicted to result in a frame shift leading to premature termination. The corresponding protein in human, retinal guanylate cyclase 1 (GUCY2D), plays an important role in rod and cone photoreceptor cell function. Loss-of-function mutations in human GUCY2D cause LCA1, one of the most common forms of Leber congenital amaurosis, which results in blindness at birth or in early childhood. The early loss of cone and reduced rod photoreceptor cell function in the cpfl9 mutant is accompanied by a later, progressive loss of cone and rod photoreceptor cells, which may be relevant to understanding disease pathology in a subset of LCA1 patients and in individuals with cone-rod dystrophy caused by recessive GUCY2D variants. cpfl9 mice will be useful for studying the role of Gucy2e in the retina.
... Current models suggest that rhodopsin and other integral membrane proteins traffic by anterograde IFT, mediated by kinesin-2, along the CC for deposition into nascent discs [61][62][63][64]. In our hands iCre75-based conditional knockout of KIF3A, the kinesin-2 obligatory subunit, did not prevent trafficking of rhodopsin and other proteins to the OS [65]. Conditional knockout of KIF3A with Six3Cre prevented CC and axoneme formation, although BB docked to the cell membrane correctly [60]. ...
... Sections were blocked in 5% normal goat serum (NGS); 0.1% Triton in 1X PBS (for 1 hour 4% PFA fixation) or 5% NGS in 1X PBS (for 10 minutes 4% PFA or 10 minutes methanol-acetone fixation) for 1 hour. Antibodies, fixation, dilutions and sources: PDE6 (1 hour 4% PFA, 1:1000; MOE Cytosignal) [64]; OPN1MW/OPN1LW (1 hour 4% PFA, 1:500, Millipore Sigma AB5405) [65]; CEP164 (10 m minutes in 4% PFA, 1:350, Sigma-Aldrich); ARL13B (1 hour 4% PFA,1:350, ProteinTech); IFT57 (10 minutes 4% PFA, 1:200, Pazour lab); IFT88 (10 minutes 4% PFA, 1:200, Pazour lab); IFT120 (10 minutes 4% PFA, 1:150, Pazour lab); SPATA7 (10 minutes 4% PFA, 1:100, Chen lab, purified as described [35]); CEP290 (10 minutes 4% PFA, 1:300, Swaroop laboratory); PCNT (10 minutes 4% PFA, 1:250, Covance); Ninein (10 minutes 4% PFA, 1:100, Abclonal and Sigma-Aldrich); NPHP1 (10 minutes 4% PFA, 1:100, [68]); FBF1 (10 minutes methanol-acetone fix, 1:200, Proteintech). Primary antibodies were diluted in blocking buffer and applied to sections; sections were then incubated overnight at 4˚C. ...
Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts ( ret Cep164 -/- ) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts ( rod Cep164 -/- ), Cre expression starts after P7 and CC/OS form. P16 rod Cep164 -/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rod Cep164 -/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rod Cep164 -/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1 ETCre ; Cep164 F/F ( tam Cep164 -/- ) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.
... In zebrafish, mutations in KIF3a caused rapid rod degeneration due to Rhodopsin accumulation (73). However, recent studies showed that KIF3a is required for the transport of proteins in the cones but not rod photoreceptor (PR) cells in mice with KIF3a depleted PR cells (74). ...
Background:
Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, wherein the perception of vision is initiated when these neurons respond to photons in the light stimuli. The photoreceptor cell is structurally studied as outer segments (OS) and inner segments (IS) where proper protein sorting, localization, and compartmentalization are critical for phototransduction, visual function, and survival. In human retinal diseases, improper protein transport to the OS or mislocalization of proteins to the IS and other cellular compartments could lead to impaired visual responses and photoreceptor cell degeneration that ultimately cause loss of visual function.
Results:
Therefore, studying and identifying mechanisms involved in facilitating and maintaining proper protein transport in photoreceptor cells would help our understanding of pathologies involving retinal cell degeneration in inherited retinal dystrophies, age-related macular degeneration, and Usher Syndrome.
Conclusions:
Our mini-review will discuss mechanisms of protein transport within photoreceptors and introduce a novel role for an unconventional motor protein, MYO1C, in actin-based motor transport of the visual chromophore Rhodopsin to the OS, in support of phototransduction and visual function.
... Although ciliary cargoes clearly accumulate at the BB with intraflagellar transport (IFT) proteins and motor proteins in rod CC, the organization of ciliary cargoes within the structures of the BB region of any primary cilium remains generally unexplored. See [7,78,133,146,166,197] for reviews of the roles of IFT proteins and motor proteins in trafficking to and from the cilium in photoreceptors and other cell types. ...
The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structure and distribution of molecular components of these structures are still incompletely understood, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.
... The retinal sections were permeabilized using 5% donkey serum in PBS containing 0.2% Triton X-100, as described previously. The sections were incubated with primary polyclonal anti-rhodopsin antibody (1 : 900, VPP, custom made [12]) and polyclonal anti-S-opsin antibody (1 : 500, Millipore, USA) at royalsocietypublishing.org/journal/rsob Open Biol. 11: 210076 ...
Immunohistochemistry (IHC) using mouse retinal cryosections is widely used to study the expression and intracellular localization of proteins in mouse retinas. Conventionally, the preparation of retinal cryosections from mice involves tissue fixation, cryoprotection, the removal of the cornea and lens, embedding and sectioning. The procedure takes 1–2 days to complete. Recently, we developed a new technique for the preparation of murine retinal cryosections by coating the sclera with a layer of Super Glue. This enables us to remove the cornea and extract the lens from the unfixed murine eye without causing the eyecup to collapse. In the present study, based on this new technique, we move a step forward to modify the conventional protocol. Unlike in the conventional protocol, in this method, we first coat the unfixed mouse eyeball on the sclera with Super Glue and then remove the cornea and lens. The eyecup is then fixed, cryoprotected and sectioned. This new protocol for the preparation of retinal cryosections reduces the time for the procedure to as little as 2 h. Importantly, the new protocol consistently improves the morphology of retinal sections as well as the image quality of IHC. Thus, this new quick protocol will be greatly beneficial to the community of visual sciences by expediting research progress and improving the results of IHC.
... In mice, photoreceptor-specific knockout of Kif3a results in a retinal phenotype very similar to the tulp1−/− phenotype including rhodopsin and arrestin mislocalization and progressive photoreceptor cell death, suggesting that Kinesin-2/IFT complexes mediate opsin transport [11,12,[52][53][54]. Conversely, a separate conditional Kif3a knockout in mouse rod photoreceptors surprisingly showed normal trafficking of rhodopsin as well as other OS membrane proteins even during rapid photoreceptor degeneration [55]. Furthermore, a retina-specific tamoxifen-inducible deletion of Kif3a in adult mice showed normal rhodopsin localization before degeneration while the same deletion in embryonic mice resulted in lack of axoneme/CC formation [34]. ...
Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.
... Conditional knockout of the kinesin motor subunit, KIF3A, in mouse photoreceptors resulted in the mislocalization of opsin to the inner segment, leading to the conclusion that IFT is essential for connecting cilium rhodopsin transport [99,129]. In contrast, transport appeared normal for 2 to 4 weeks in a rod-specific Kif3A knockout mouse [6]. Similarly, rhodopsin localization was normal for 2 weeks after retina-specific tamoxifen-inducible deletion of KIF3A and IFT88 in adult mice using the six3 Cre driver. ...
... Interestingly, cone-specific Kif3A knockout resulted in major mislocalization of phototransduction components in cones [6] and expression of a dominant negative Kif3B mutant in Zebrafish results in accumulation of large vesicles in cone IS and disruption of OS lamellar morphogenesis [93]. Finally, mutations in Bardet-Biedl complex (BBSome) proteins cause mislocalization of cone opsin and phototransduction proteins [1,11]. ...
Retinal photoreceptors are neurons that convert dynamically changing patterns of light into electrical signals that are processed by retinal interneurons and ultimately transmitted to vision centers in the brain. They represent the essential first step in seeing without which the remainder of the visual system is rendered moot. To support this role, the major functions of photoreceptors are segregated into three main specialized compartments—the outer segment, the inner segment, and the pre-synaptic terminal. This compartmentalization is crucial for photoreceptor function—disruption leads to devastating blinding diseases for which therapies remain elusive. In this review, we examine the current understanding of the molecular and physical mechanisms underlying photoreceptor functional compartmentalization and highlight areas where significant knowledge gaps remain.
... Both are synthesized in the cytosol, dock to the ER for posttranslational processing (isoprenylation) and traffic to the OS in a complex with PDE6D by diffusion, independent of dynein [27]. Using anti-PDE6 antibody (MOE) that recognizes both rod and cone PDE6 [28], we show that PDE6 localizes exclusively to OS of control cryosections (Fig 4, row C). Iin P6 mutant sections, PDE6 accumulates together with centrosomes in a cytosolic space or in foci at the sclerad edge of the ONL (Fig 4, row B). ...
Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1 F/F ;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1 F/F ;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1 F/F ;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration.
... KIF3A conditionally knocked out in mouse photoreceptors resulted in the mislocalization of opsin, leading to the conclusion that KIF3A, and by extension IFT, is essential for rhodopsin transport within the CC (Marszalek et al., 2000;Jimeno et al., 2006). In contrast, in another rod specific KIF3A knockout mouse rhodopsin and many of the other phototransduction proteins appeared to transport normally to the OS for 2-4 weeks, after which rods degenerated (Avasthi et al., 2009). Similarly, retina specific tamoxifen inducible deletion of KIF3A and IFT88 in adult mice showed normal localization of rhodopsin for 2 weeks before degeneration of photoreceptors while the same deletion from embryonic stage resulted in lack of assembly of the CC (Jiang et al., 2015b). ...
... Interestingly, there was major mislocalization of phototransduction components in cone specific KIF3A knockout mice (Avasthi et al., 2009), and KIF3B dominant negative mutant causes accumulation of large vesicles in cone IS and disruption of cone OS morphogenesis . These results suggest that IFT is required for transport of opsin and other phototransduction proteins in cones, but not in rods. ...
Functional compartmentalization of cells is a universal strategy for segregating processes that require specific components, undergo regulation by modulating concentrations of those components, or that would be detrimental to other processes. Primary cilia are hair-like organelles that project from the apical plasma membranes of epithelial cells where they serve as exclusive compartments for sensing physical and chemical signals in the environment. As such, molecules involved in signal transduction are enriched within cilia and regulating their ciliary concentrations allows adaptation to the environmental stimuli. The highly efficient organization of primary cilia has been co-opted by major sensory neurons, olfactory cells and the photoreceptor neurons that underlie vision. The mechanisms underlying compartmentalization of cilia are an area of intense current research. Recent findings have revealed similarities and differences in molecular mechanisms of ciliary protein enrichment and its regulation among primary cilia and sensory cilia. Here we discuss the physiological demands on photoreceptors that have driven their evolution into neurons that rely on a highly specialized cilium for signaling changes in light intensity. We explore what is known and what is not known about how that specialization appears to have driven unique mechanisms for photoreceptor protein and membrane compartmentalization.
... If I started over in science 1000 times, 999 of those times, I think I would end up studying the cytoskeleton. As a graduate student at the University of Utah in Wolfgang Baehr's lab, I studied trafficking through photoreceptor cilia in mice (Avasthi et al., 2009(Avasthi et al., , 2013Ying et al., 2014) and realized through my reading that so many of the fundamental discoveries in this field were made in the unicellular green alga Chlamydomonas. The idea of working in a model system that allowed mechanistic analyses and discovery of new regulatory pathways was extremely appealing, particularly a system so well suited to biochemical and genetic approaches. ...
I always found it curious that in science, we value unique, creative thinkers, but we teach scientists to progress in a formulaic manner that rarely takes each person’s individual strengths into account. Surprisingly, when we break the mold, we are often rewarded for it. This cycle of learning to survive using conventional wisdom but being rewarded for a unique path outside of it seems to be an unspoken key to success. I am honored to be awarded the 2020 Women in Cell Biology Junior Award for Excellence in Research and am thrilled to share some of the unconventional guiding principles that brought me to where I am in this rich scientific landscape. The game changers in the early phase of my career were informal mentors, open scientific communication, and persistence in pursuing difficult scientific questions.
