Intrinsic heterogeneity of primary cilia revealed through spatial proteomics.

The Emerging Complexity of the Vertebrate Cilium: New Functional Roles for an Ancient Organelle

Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

The precise trafficking and spatial organization of signaling molecules within cells is critical for many fundamental cellular processes. Two interconnected microtubule-based organelles, the centrosome and primary cilium, have been making headlines recently due to their role as central “hubs” for coordinating such signaling events. The centrosome is the major microtubule-nucleating center in animal cells, which polarizes microtubule arrays and thereby directs microtubule-based trafficking toward itself and its associated structure, the primary cilium. [1]. The primary cilium is a tiny hair-like sensory organelle that is templated by one of two centrioles, core elements of the centrosome, and protrudes above the apical surface of almost every cell in the human body (Figure 1). Together, the centrosome and cilium mediate the initiation and transmission of extracellular signals to the interior of the cell, thus controlling many aspects of cell physiology [2], [3]. Defects in the structure and/or function of these organelles result in human disease conditions termed “ciliopathies,” a heterogeneous group of disorders with phenotypes including cystic kidneys; digit, bone, and brain anomalies; infertility; and even cancer [4], [5].

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

RTTN Mutations Link Primary Cilia Function to Organization of the Human Cerebral Cortex

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.

Background: The Hedgehog (HH) pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle not expressed in the normal colonic epithelium which recruits the functional components and regulates the pathway. Aims: Since the intestinal epithelium is known to be a non-ciliated tissue, the HH pathway as related to the PC has not been explored. We investigated the presence of PC in colon cancer tumors and cell lines. Our hypothesis is that PC formation controls the HH activation that has been reported in numerous colon cancer studies. Materials and methods: We used cellular models to look at HH activation, focusing on the final effector Gli1. The link between PC and the HH pathway was shown by the recruitment of the Smo receptor in the PC. We looked for PC in a subset of 63 tumors from a Tissue Micro Array and in 4 colorectal cell lines by immunofluorescence using two well-known markers, acetylated α-tubulin and polyglutamylated tubulin. 3D deconvoluted pictures were obtained to characterize the shape and size of PC. Using cellular models we investigated HH pathway expression by qPCR. We assessed the functional link between HH pathway and PC through localization of the Smo receptor in the PC using immunofluorescence. Results: We observed the presence of the PC in the epithelium of primary colorectal tumors at all stages but not in their normal counterparts. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the PC membrane. Conclusion: We conclude that the PC directly participates in the HH pathway in colorectal cancer cells which is the first observation of PC expression in tumors arising from a non-ciliated tissue. Functional studies are required to determine the mode of action of this organelle in carcinogenesis. Citation Format: Blanche Senicourt. Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4430.

Most renal tubular epithelial cells possess primary cilia that protrude into the tubular lumen, where they are exposed to urine flow. The cilia of the epithelia curve with the urine flow within the renal tubules. The primary cilium of renal epithelium has been assumed to be a mechano-sensor of the urine flow. However, the mechanical characteristics of the cilium of an each cell are not well assessed. In this study, we measured Young's modulus of each primary cilium by using optical tweezers. First, we developed a system to manipulate each primary cilium of living tubular epithelial cells in situ by using optical tweezers. We adopted flexible-substratum technique to visualize individual micron-sized primary cilia of the Madin-Darby canine kidney (MDCK) cell lines. Through the use of the flexible-substratum technique, the primary cilia of MDCK cells could be clearly imaged from the side. We showed that we could apply minute force to polystyrene microspheres stuck on the tip of the cilia by using optical tweezers. The Young's modulus of each primary cilium was successfully examined by measuring the displacement of the microsphere. We concluded that this methodology enables us to manipulate each primary cilium minutely and to estimate the elasticity of an individual primary cilium.

Motile and immotile (or primary) cilia are microtubule-based structures that mediate multiple cellular functions, including the transduction of environmental cues, developmental signaling, cellular motility, and modulation of fluid flow. Although their core architectures are similar, motile and primary cilia exhibit marked structural differences that underlie distinct functional properties. However, the extent to which ciliogenesis mechanisms are shared between these different cilia types is not fully described. Here, we report that the atypical MAP kinase MAPK15 (ERK7/8), implicated in the formation of vertebrate motile cilia, also regulates the formation of primary cilia in Caenorhabditis elegans sensory neurons and human cells. We find that MAPK15 localizes to a basal body subdomain with the ciliopathy protein BBS7 and to cell-cell junctions. MAPK15 also regulates the localization of ciliary proteins involved in cilium structure, transport, and signaling. Our results describe a primary cilia-related role for this poorly studied member of the MAPK family in vivo, and indicate a broad requirement for MAPK15 in the formation of multiple ciliary classes across species.

Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

Fluid intake is tightly related to blood pressure control through balance between electrolytes and water in the body. Virtually, every mammalian cell is equipped with a primary cilium, a cell surface protrusion that is thought to act as a sensory organelle. However, the contribution of primary neuronal cilia to cardiovascular regulation and fluid homeostasis has not been addressed. We hypothesized that primary neuronal cilia contribute to the control of fluid intake and blood pressure. For this, we first examined the length of primary neuronal cilia in the brain nuclei that contribute to osmolarity and cardiovascular regulation in 3‐week DOCA‐salt treated mice vs sham controls (n=5/group). Cilia length was examined by immunostaining a ciliary protein, adenylate cyclase 3 (AC3), followed by confocal 3D reconstruction, and quantification by IMARIS imaging analysis software. We found changes in cilia length in a number of brain nuclei of DOCA‐salt mice. The supraoptic nucleus (SON), a key brain nucleus in the regulation of osmolarity and fluid homeostasis, displayed the most significant changes in cilia length (average primary cilia: 7.46±□□□□□m in DOCA‐salt mice vs 6.76±□□□□□m in sham, p=0.0509). Moreover, the number of primary cilia that are over 10□□m was significantly increased in the SON of DOCA‐salt mice by 8% (p=0.0114). On the other hand, the number of cilia that are 4–5□□m in length was significantly decreased in the SON of DOCA‐salt mice (11.73±1.70%) compared to sham controls (18.73±2.02%, p=0.0385). Next, we assessed the relevance of SON cilia by targeting the ift88 gene which encodes a protein necessary for the formation of cilia. For this, we performed targeted stereotaxic injection of AAV‐Cre into the SON of IFT88flox/flox mice. We found that disruption of SON ift88 (AAV‐Cre group had a 38% ift88 expression level relative to AAV‐GFP control group) caused impairment in water intake in response to 24h water deprivation (30min water intake: 0.78±0.06 ml in AAV‐Cre vs 1.07±0.08 ml in AAV‐GFP, p=0.0129) without affecting total daily water and food consumption. Moreover, ift88 deletion in SON increased systolic blood pressure (133.7±1.71 mmHg vs 115.8±2.33 in AAV‐GFP, p=0.0008) without altering heart rate. Our data demonstrate the importance of primary cilia in the SON in the regulation of water intake and blood pressure. We speculate that primary neuronal cilia may function as part of environmental surveillance system in the brain that ensures cardiovascular homeostasis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rat foetuses from intra-uterine days E13 through E22 (day before parturition) and adults were used for a qualitative electron-microscopic investigation of the development of ciliated/microvillous surfaces of the olfactory epithelium. In the E13 and most of the E14 embryos the epithelial surface is not yet characteristically olfactory. Apical cell profiles show primary cilia. These can arise at the epithelial surface or below. From E14 onwards the epithelial surface acquires olfactory characteristics. Dendritic endings of the olfactory receptor cells can be found amidst microvillous profiles of supporting cells. Either cell type may bear primary cilia. From E16 onwards the receptor cells sprout multiple olfactory cilia, but cells with primary cilia are found throughout pre-natal development. These primary cilia are, at least for a while, retained during the formation of the secondary cilia. Primary cilia always have distinct necklaces at their base. Otherwise, especially with respect to their tips, their morphology can vary. Originally they have expanded tips (up to E14); later on such wide tips are no longer encountered (E16 and E17). Primary cilia of receptor cells never have wide tips. Appreciable numbers of endings with tapering olfactory cilia are discerned around E18 and especially E19. Throughout pre-natal development posterior/superior parts of the septal olfactory epithelium are more precocious than anterior/inferior parts, in particular in the region of transition with the respiratory epithelium. This advance in development includes total densities of dendritic endings of olfactory receptor cells, densities of multiciliated endings alone and lengths of supporting cell microvilli. This difference is discussed with respect to the topography of the olfactory epithelial surface in adult animals. In addition to the systematic topographic variation, a number of more local, apparently not-systematically distributed, topographic variations present during development are described. Most of these also occur in adult animals and they include heterogeneity in length of supporting cell microvilli and the presence of patches of supporting cells with rounded apical protuberances, of patches displaying dendrites with polyaxonemes rather than individual cilia and of scattered atypical cells (neither typical olfactory receptor nor olfactory supporting cells). At their surfaces such atypical cells can resemble inner-ear hair cells. Relative to olfactory receptor and supporting cells there are only very few atypical cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Modeling Human Disease in Humans: The Ciliopathies

Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1, PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 micron long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PK1 exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0.05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.