Specialized Cilia in Mammalian Sensory Systems

Abstract

Cilia and flagella are highly conserved and important microtubule-based organelles that project from the surface of eukaryotic cells and act as antennae to sense extracellular signals. Moreover, cilia have emerged as key players in numerous physiological, developmental, and sensory processes such as hearing, olfaction, and photoreception. Genetic defects in ciliary proteins responsible for cilia formation, maintenance, or function underlie a wide array of human diseases like deafness, anosmia, and retinal degeneration in sensory systems. Impairment of more than one sensory organ results in numerous syndromic ciliary disorders like the autosomal recessive genetic diseases Bardet-Biedl and Usher syndrome. Here we describe the structure and distinct functional roles of cilia in sensory organs like the inner ear, the olfactory epithelium, and the retina of the mouse. The spectrum of ciliary function in fundamental cellular processes highlights the importance of elucidating ciliopathy-related proteins in order to find novel potential therapies.

Keywords: connecting cilium; inner ear; intraflagellar transport; kinocilium; olfactory epithelium; primary cilia; retina.

Figure 1

The primary cilium and its intraflagellar transport machinery. (A–D) Triple labeling of acetylated α-tubulin (A green, marker for the ciliary axoneme), ADP-ribosylation factor-like 13B (Arl13B; B red, marker for the ciliary membrane) and DAPI (D blue) as a nuclear marker in serum-starved NIH 3T3 mouse fibroblasts; (C,D) Merged images showing the localization of acetylated α-tubulin and Arl13B at a single primary cilium projecting from the cell surface; (E) 3D reconstruction of the non-motile primary cilium demonstrating that Arl13B (red) ensheathes the axoneme of the primary cilium (PC) labeled with an antibody against acetylated α-tubulin (green); (F) Schematic of the primary cilium and its intraflagellar transport machinery. The primary cilium is divided into the basal body complex (shaded in blue) and the membrane-bound axoneme (9 × 2 + 0 microtubule configuration) extending from the surface. The basal body complex comprises the basal body and its centriole (9 × 3 + 0 structure) enclosed by the pericentriolar material (PCM). Elongation of the axoneme is mediated by the intraflagellar transport (IFT) machinery. Anterograde IFT from the base to the tip of the cilium depends on IFT B proteins and the microtubule plus-end-directed kinesin II motor protein family. Retrograde IFT from the tip to the base of the cilium depends on IFT A proteins and the minus-end-directed cytoplasmic dynein 2 motor protein. Scale bars: 5 µm (A–D), 2 µm (E).

Figure 2

Cilia in the inner ear of mammals shortly after birth. (A,B) Cross section of a mouse cochlea at P5 (postnatal day 5) stained with toluidine blue. The cochlea is divided into three fluid-filled compartments: scala vestibuli, scala tympani and scala media. Scala vestibuli (containing perilymph) is separated from the scala media (containing endolymph) by the Reissner’s membrane, while scala media and scala tympani (containing perilymph) are separated by the basilar membrane. The basilar membrane contains the Organ of Corti with sensory hair cells responsible for auditory function. The hair cells are arranged in four rows along the entire length of the cochlea—three rows of outer and one row of inner hair cells. The tectorial membrane responsible for their direct (outer hair cells) and indirect (inner hair cells) activation covers the hair cells; (C) Schematic of a mammalian cochlear hair cell shortly after birth. Hair cells are polarized epithelial mechanosensory cells with a mechanically sensitive organelle at the apical surface, known as stereovilli (SV). Stereovilli are non-motile hair bundles consisting of dozens of specialized F-actin-filled microvilli graduated in length to form a staircase-like structure. Within their V-shaped orientation stereovilli are connected by extracellular linkages called tip links. The longest stereovilli is closest to a single genuine microtubule-based cilium, the kinocilium with a (9 × 2 + 2) microtubule configuration (which begins to regress at around P8 in the mouse). Hair cells are surrounded by non-sensory supporting cells with microvilli on their apical surface; (D–H) Transmission electron micrographs of a mouse cochlea; (D) Cross-section of a kinocilium (KC) showing the (9 × 2 + 2) structure; (E) Longitudinal section of stereovilli and the kinocilium on the apical surface of an inner hair cell; (F) Immature synaptic ribbon (SR) of an inner hair cell with the typical electron-dense sphere surrounded by synaptic vesicles; (G) V-shaped orientation of stereovilli and the kinocilium on a vestibular hair cell; (H) Cross-section of a synaptic ribbon in a vestibular hair cell. CS: centrosome. CP: cuticular plate. Scale bars: 50 µm (A,B), 100 nm (D,H), 200 nm (E,F), 500 nm (G).

Figure 3

Structure of cilia in the mouse respiratory and olfactory epithelium. (A–C) Transmission electron micrographs of an adult mouse olfactory epithelium; (A) The respiratory epithelium contains multiple motile cilia on one cell; (B) Higher magnification of a cross section showing the (9 × 2 + 2) microtubule configuration of olfactory cilia; (C) Longitudinal section of an olfactory knob (OK) with extending olfactory cilia (OC); (D) Schematics of an olfactory sensory neuron (OSN) and its olfactory knob. OSNs are the receptor elements of the olfactory system. They are surrounded by supporting cells (SC) with a microvilli (MV) border on their apical surface and continually replaced by basal cells (BC) throughout life. OSNs are bipolar neurons with dendrites ending in an olfactory knob which has specialized sensory cilia responsible for olfaction. The mammalian olfactory cilium comprises the transition zone (TZ), the proximal segment (PS), and the distal segment (DS). The TZ (9 × 2 + 0 structure) is located at the base of the olfactory cilium between the basal body and the origin of the axoneme’s central pair of microtubules. The PS projects from the basal body in a (9 × 2 + 2) configuration. The DS represents the end of the cilium and contains characteristic arrays of singlet microtubules (from 9 × 1 to 2 × 1); (E–H) Higher magnification electron micrographs showing the different microtubule configurations of the DS (E–G) and PS (H) of an olfactory cilium. CS: centrosomes. BL: basal lamina. Scale bars: 500 nm (A,C), 200 nm (B), 100 nm (E–H).

Figure 4

Immunofluorescence analysis of olfactory cilia in the mouse olfactory epithelium. (A–C) Confocal laser scanning micrographs of a cryostat section through adult mouse olfactory epithelium double labeled with antibodies against Centrin 3 (green) and polyglutamylated tubulin (red); (A,B) Centrin 3 and polyglutamylated tubulin are localized in the ciliary region of the olfactory sensory neurons (OSNs); (C) Merge combined with a DAPI nuclear staining (blue); (D–F) Higher magnification views of the ciliary region; (D) Centrin 3 is preferentially localized at the basal body, the centriole and the transition zone of cilia in the olfactory knobs (OK) of receptor neurons; (E) Polyglutamylated tubulin labels the axoneme of olfactory cilia; (F) The merge shows a partial colocalization of Centrin 3 and polyglutamylated tubulin at the transition zone of the olfactory receptor neurons. ML: mucus layer. Scale bars: 10 µm (C for A–C), 1 µm (F for D–F).

Figure 5

Organization of rod photoreceptors in the mammalian retina. (A) Vertical section of a vertebrate retina stained with toluidine blue. The retina contains five classes of neurons (photoreceptors, bipolar cells, horizontal cells, amacrine cells, ganglion cells), and is divided into three nuclear layers (outer nuclear layer (ONL), inner nuclear layer (INL), ganglion cell layer (GCL)) and two synaptic layers (outer plexiform layer (OPL), inner plexiform layer (IPL)); (B) Schematic of a vertebrate rod photoreceptor consisting of the long, light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE) which is connected via a small intracellular bridge, the connecting cilium (CC), to the metabolically active inner segment (IS) including the basal body complex (BBC; shaded in blue) and the apical region. The OS represents a modified primary cilium. The CC corresponds to the transition zone of a prototypic cilium with a (9 × 2 + 0) microtubule array; (C–H) Transmission electron micrograph of a photoreceptor-connecting cilium; (C) The CC connects the OS and IS. The BBC consists of the basal body (BB) and its centriole (Ce); (D) Cross-section of the CC showing the characteristic (9 × 2 + 0) microtubule array; (E) RPE with pigment granules and the Bruch’s membrane (BM); (F) OS composed of stacks of tightly packed membrane discs; (G) Higher magnification view of the ciliary apparatus; (H) Synaptic ribbon (SR) with invaginations of postsynaptic horizontal cells (HC) and bipolar cells (BC). Scale bars: 100 µm (A), 200 nm (D,F,H), 500 nm (E,G).

Figure 6

The connecting cilium of photoreceptors and its transport machinery. (A) Schematic of a vertebrate rod photoreceptor consisting of the light-sensitive outer segment (OS) enclosed by the retinal pigment epithelium (RPE) and linked via the connecting cilium (CC) to the metabolically active inner segment (IS); (B,C) Schematics of the two ciliary transport systems in photoreceptors; (B) Cargo transport to the CC is mediated along microtubules (MT) by cytoplasmic dynein 1 motor proteins (minus-end-directed). Delivery from the IS to the OS is mediated by IFT B molecules (binding the cargo) and kinesin II motor proteins (plus-end-directed). Delivery back to the IS is mediated by IFT A molecules and cytoplasmic dynein 2 motor proteins (minus-end-directed); (C) In addition, a myosin 7a-driven transport along actin filaments is used for trafficking proteins to the OS; (D–G) Confocal laser scanning micrographs of a vertical cryostat section through an adult mouse retina double labeled for Centrin 3 (D, green) and Pericentrin (E, red) as markers for the cilia and the basal body complex (BBC), respectively; (F,G) As seen in the merge of the stainings with additional labeling of the cell nuclei with DAPI, centrin 3 and pericentrin partially colocalize at the ciliary region of the photoreceptors and at the centrosomes of other retinal cells; (H–J) Higher power views showing the partial colocalization of centrin 3 and pericentrin at the BBC of photoreceptor-connecting cilia. Scale bars: 20 µm (F), 1 µm (J).