Article Figures & Data
Figures
- Fig. 1.
Microtubule network in a rod photoreceptor. One pathway delivers material toward the outer segment along the connecting cilium (CC) axoneme, the other carries Golgi-derived vesicles to the synaptic terminal along cytoplasmic microtubules.Arrows indicate microtubule (mt) polarity. Brackets show the two sites of KIF3A localization: the basal body (BB) of the connecting cilium axoneme, and the synaptic ribbon.
- Fig. 2.
KIF3 motors are expressed in the rat retina.A, A retina homogenate from adult animals was analyzed by Western blotting with antibodies to conventional kinesin heavy chain (KHC), KIF3A, KIF3B, KIF3C, and KAP3A. Similar to brain KIF3A (Kondo et al., 1994; Muresan et al., 1998), retinal KIF3A is detected as a doublet, with the upper band being predominant.B, Expression of KIF3A and KHC in neonatal retina.P0, P7, and P35 indicate day 0, 7, and 35 after birth. Note that the level of KIF3A, but not KHC, increases during postnatal development of the retina.C, Nucleotide-dependent microtubule binding and release of retinal kinesins. A Triton X-100-solubilized retinal homogenate was incubated with microtubules and AMP-PNP. Microtubules were separated from the depleted cytosol and extracted with ATP and NaCl. Supernatant (S) and pellet (P) of extracted microtubules were obtained by centrifugation.
- Fig. 3.
KIF3A, KIF3C, and conventional kinesin (KHC) show distinct distributions in the rat retina. Fixed-frozen sections of rat eyecups were processed for immunocytochemistry with antibodies to KIF3C, KIF3A, and KHC, or a preimmune IgG fraction from the rabbit immunized with KIF3C. Two corresponding phase-contrast micrographs are also shown. Note that antibodies to the kinesin polypeptides label different cell types in different layers of the retina. Also note that antibodies to KIF3A and KIF3C label different structures in the OPL. RPE, Retinal pigment epithelium; OIS, outer and inner segments of photoreceptor cells; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer;GCL, ganglion cell and optic nerve fiber layers. Scale bar, 50 μm.
- Fig. 4.
The distribution of KIF3A is included within the distribution of synaptic vesicle markers. Fixed-frozen sections through rat retina were double-stained with anti-KIF3A antibody and either synaptotagmin (A) or CSP1 (B). KIF3A was detected with fluorescein isothiocyanate-labeled secondary antibodies (green), whereas rhodamine-labeled secondary antibodies (red) were used to detect the synaptic vesicle markers. The merged images were obtained by simultaneous visualization of both fluorophores through a broad-band filter. Note the discrete distribution of KIF3A as opposed to the continuous distribution of the synaptic vesicle markers. The fluorescein isothiocyanate-labeled secondary (anti-mouse IgG) antibody used in these experiments cross-reacts with rat IgG and labels capillaries (arrows, B). Only the OPL is shown inB. Scale bar, 20 μm.
- Fig. 5.
KIF3A is localized to the ribbon synapses of retinal photoreceptors. Fixed-frozen sections from rat retinas were processed for immunofluorescence microscopy with anti-KIF3A antibody (A, B). The shape of the mammalian photoreceptor ribbon, shown in the three-dimensional model in C, explains the horseshoe-like appearance of the anti-KIF3A-labeled structures at high magnification (B). Note that in the OPL (A, top image), the labeled structures appear either clustered (typical for cone terminals; arrows) or as individual structures (typical for rod synapses). The bottom image in A is from the IPL. The three-dimensional model in C (modified fromRao-Mirotznik et al., 1995) shows the ribbon of the presynaptic photoreceptor cell with the docked vesicles and the postsynaptic horizontal (Hz) and bipolar (Bp) cells. Scale bars: A, 25 μm; B, 5 μm.
- Fig. 6.
KIF3A is detected at the synaptic ribbon of photoreceptor synapses. Transmission electron micrographs from rat retinal specimens processed for postembedding immunocytochemistry (A, B) or cryoimmunoelectron microscopy (C, D) with anti-KIF3A antibodies. Gold particles are localized to vesicular organelles associated with the synaptic ribbons and scattered throughout the cytoplasm. The highest labeling density is detected over and around the ribbons themselves. Numerous vesicles at the synaptic terminal are unlabeled (B, arrow). The plane of section in B traverses both the presynaptic photoreceptor cell and the postsynaptic horizontal or bipolar cell.Arrowheads show the postsynaptic membrane.
- Fig. 7.
Detergent-soluble and insoluble fractions of KIF3A. A, B, Rat retina immunocytochemistry. Fresh-frozen sections were extracted with Triton X-100 (B) or buffer (A,control) before formaldehyde fixation and staining with antibodies to KIF3A and CSP1. Bottom images in B are corresponding phase-contrast micrographs. Scale bar, 50 μm. C, Subcellular fractionation of rat retina. A crude rat retina synaptic membrane fraction was extracted with Triton X-100. After centrifugation, the supernatant (S) and pellet (P) were analyzed by Western blotting with antibodies to KIF3A and CSP1.
- Fig. 8.
KIF3A is localized to the photoreceptor axoneme.A, B, Rat retina immunocytochemistry. Fresh-frozen sections were extracted with Triton X-100, fixed with formaldehyde, and stained with anti-KIF3A antibody. Note that photoreceptor outer segments have been solubilized by detergent extraction. Some extracted axonemes are indicated byarrows (B). The location of thelabeled dots in A corresponds to the base of the axonemes (black arrow). B is a phase-contrast micrograph corresponding to A. Scale bar, 20 μm. C, Rod inner and outer segments (RIS-ROS) contain KIF3A and KAP3A. RIS-ROS were prepared from rat retina and analyzed by Western blotting. D, Western blotting of a bovine photoreceptor axoneme fraction with antibodies to KIF3A, KIF3B, KIF3C, and KAP3A.
