Ciliary Length Research Articles

Ciliogenesis: building the cell's antenna

The cilium is a complex organelle, the assembly of which requires the coordination of motor-driven intraflagellar transport (IFT), membrane trafficking and selective import of cilium-specific proteins through a barrier at the ciliary transition zone. Recent findings provide insights into how cilia assemble and disassemble in synchrony with the cell cycle and how the balance of ciliary assembly and disassembly determines the steady-state ciliary length, with the inherent length-dependence of IFT rendering the ciliary assembly rate a decreasing function of length. As cilia are important in sensing and processing developmental signals and directing the flow of fluids such as mucus, defects in ciliogenesis and length control are likely to underlie a range of cilium-related human diseases.

Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry

The primary cilium is an antenna-like organelle that is dynamically regulated during the cell cycle. Ciliogenesis is initiated as cells enter quiescence, while cilium resorption precedes mitosis. The mechanisms coordinating ciliogenesis with the cell cycle are unknown. Here we identify the centrosomal protein, Nde1, as a negative regulator of ciliary length. Nde1 is expressed at high levels in mitosis, low levels in quiescence and localizes at the mother centriole, which nucleates the primary cilium. Cells depleted of Nde1 show longer cilia and a delay in cell cycle re-entry that correlates with ciliary length. Knockdown of Nde1 in zebrafish embryos results in increased ciliary length, suppression of cell division, reduction of the number of cells forming the Kupffer’s vesicle, and left-right patterning defects. These data suggest that Nde1 is an integral component of a network coordinating ciliary length with cell cycle progression and have implications in the transition from quiescence to a proliferative state.

A cell‐based screen for inhibitors of flagella‐driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

Soluble levels of cytosolic tubulin regulate ciliary length control

The primary cilium is an evolutionarily conserved dynamic organelle important for regulating numerous signaling pathways, and, as such, mutations disrupting ciliogenesis result in a variety of developmental abnormalities and postnatal disorders. The length of the cilium is regulated by the cell through largely unknown mechanisms. Normal cilia length is important, as either shortened or elongated cilia have been associated with disease and developmental defects. Here we explore the importance of cytoskeletal dynamics in regulating cilia length. Using pharmacological approaches in different cell types, we demonstrate that actin depolymerization or stabilization and protein kinase A activation result in a rapid elongation of the primary cilium. The effects of pharmacological agents on cilia length are associated with a subsequent increase in soluble tubulin levels and can be impaired by depletion of soluble tubulin with taxol. In addition, subtle nocodazole treatment was able to induce ciliogenesis under conditions in which cilia are not normally formed and also increases cilia length on cells that have already established cilia. Together these data indicate that cilia length can be regulated through changes in either the actin or microtubule network and implicate a possible role for soluble tubulin levels in cilia length control.

Spatial Distribution of Calcium-Gated Chloride Channels in Olfactory Cilia

BackgroundIn vertebrate olfactory receptor neurons, sensory cilia transduce odor stimuli into changes in neuronal membrane potential. The voltage changes are primarily caused by the sequential openings of two types of channel: a cyclic-nucleotide-gated (CNG) cationic channel and a calcium-gated chloride channel. In frog, the cilia are 25 to 200 µm in length, so the spatial distributions of the channels may be an important determinant of odor sensitivity.Principal FindingsTo determine the spatial distribution of the chloride channels, we recorded from single cilia as calcium was allowed to diffuse down the length of the cilium and activate the channels. A computational model of this experiment allowed an estimate of the spatial distribution of the chloride channels. On average, the channels were concentrated in a narrow band centered at a distance of 29% of the ciliary length, measured from the base of the cilium. This matches the location of the CNG channels determined previously. This non-uniform distribution of transduction proteins is consistent with similar findings in other cilia.ConclusionsOn average, the two types of olfactory transduction channel are concentrated in the same region of the cilium. This may contribute to the efficient detection of weak stimuli.

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.

Ciliary behaviour and mechano-transduction in the embryonic node: Computational testing of hypotheses

Left–right symmetry breaking in the mammalian embryo is believed to occur in a transient embryonic structure, the node: rotational motion of cilia within this structure creates a leftward flow of liquid that is the first asymmetric event observed. A hypothesis, often referred to as the “two-cilia” hypothesis, proposes that the node contains two kinds of primary cilia: motile cilia, driven by motor proteins, that rotate clockwise generating the leftward flow and passive cilia that act as mechano-sensors, reacting mechanically to the emerging flow. The exact mechanism that underlies the initial breaking of symmetry remains unclear, in spite of several studies that have attempted to elucidate the processes involved. In this paper, we present two computational models to (i) simulate the unidirectional flow induced by the active ciliary motion as well as their propulsion on the passive cilia and to (ii) investigate the protein activity that produces the active ciliary rotation-like movement. The models presented incorporate methodologies from computational fluid dynamics, deformable mesh computational techniques and fluid–structure interaction analysis. By solving the three-dimensional unsteady transport equations, with suitable boundary conditions, we confirm that the whirling motion of active cilia is capable of inducing the unidirectional flow and that the passive cilia are pushed by this flow towards the left with a visible deformation of 41.7% of the ciliary length on the tip, supporting the plausibility of the two-cilia hypothesis. Further, by applying finite element analysis and grid deformation techniques, we investigate the ciliary motion triggered by the activation of protein motors and propose a possible dynein activation pattern that is able to produce the clockwise rotation of embryonic cilia.

Notch signalling regulates left-right asymmetry through ciliary length control

The importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer's vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer's vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture (‘mosaic’) of normal lining (EXT+/− or EXTwt/wt) and EXT−/− cells in the cartilaginous cap of osteochondromas.

Flagella and Cilia: The Long and the Short of It

Intraflagellar transport is essential for the assembly and function of cilia and flagella, and recent work shows that intraflagellar transport complexes - or trains - fall into two classes according to length and morphology. How might intraflagellar transport and the size of the trains be involved in flagellar and ciliary length control?

Zebrafish inositol polyphosphate kinases: New effectors of cilia and developmental signaling

We described here our recent findings that Ipk1 catalyzed production of IP6 regulates LR-axis specification (Sarmah et al., 2005) and that IP6 is an essential effector of ciliary beating and length maintenance in zebrafish (Sarmah et al., 2007). We have also uncovered a novel role for the IP kinase IP6k2 in craniofacial development, neural crest cell migration, and hedgehog signal transduction (B.S. an S.R.W., unpublished). Together, these findings place IP production as a key mediator for cellular signaling mechanisms that regulate vital cellular and developmental processes. How these and other IPs are integrated with cell-cell signaling networks during complex processes, such as, tissue morphogenesis and maintenance of cell fate and function? We propose that with its enormous resource and unique set of structural, functional, and sensory attributes, cilium provide a platform for executing IP-based signaling functions. Given the evolutionary conservation of the IP repertoire and pathways, the developmental and molecular events uncovered in our studies in the zebrafish system could be applicable in other vertebrates including humans. This unbiased approach of systematic identification of IP functions in cilia and development will aid in understanding of multiple disease pathologies including ciliopathies and dysmorphic syndromes.

Role of ultrasound and biomicroscopy in evaluation of anterior segment anatomy in congenital and developmental cataract cases

To investigate the role of ultrasound biomicroscopy (UBM) in the evaluation of anterior segment anatomy in cases of congenital and developmental cataract. Alexandria Main University Hospital, Ophthalmology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. In this cross-sectional nonrandomized unmasked study, ultrasound biomicroscopy (UBM) was used to evaluate the anterior segment anatomy of 32 eyes of 21 children with congenital and developmental cataract. The parents were questioned for details of the children's history. An initial office examination was done to detect visual function, pupil color, lens morphology, corneal clarity, and presence of gross anterior segment anomaly. If the fundus was visible, it was examined in cooperative children after mydriasis. Under general anesthesia, each child was examined by UBM. The parameters detected by UBM included anterior chamber depth, lens thickness, zonular fiber length, ciliary process length, anterior chamber angle, and lens morphology. A-scan biometry was also performed to detect anterior chamber biometric characteristics. Ultrasound biomicroscopy is a valuable tool for evaluating childhood cataracts and associated ocular anomalies as well as anterior segment biometric characteristics.

Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li 2CO 3 were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

16-P010 A novel role for notch signalling in left–right determination through ciliary length control

16-P010 A novel role for notch signalling in left–right determination through ciliary length control

Dynein‐2 and ciliogenesis in Tetrahymena

Dynein-2 is the motor responsible for retrograde intraflagellar transport. In situ, dynein-2 comprises four subunits: the dynein-2 heavy chain (DYH2); the dynein-2 intermediate chain; the dynein-2 light-intermediate chain (D2LIC); and dynein light chain 8 (Rompolas et al. 2007. Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor. J Cell Sci 120:3653-3665). In contrast to what has been reported in other model organisms, when the DYH2 gene or the D2LIC gene was disrupted in Tetrahymena, the cells continued to produce motile cilia that were not swollen or filled with material [Rajagopalan et al.2009. Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila. Mol Biol Cell 20:708-720]. When compared to wildtype cells, the dynein-2 mutants were found to have cilia that were at a lower density, shorter, and much more variable in length. One possible explanation for these effects is that the dynein-2 knockout cells grow cilia too slowly to enable them to achieve normal length and density before the cell divides. In the present study, dynein-2 knockout cells were deciliated and then allowed to regrow their cilia for 22 hr under conditions in which the cells did not divide. When dynein-2 was disabled, three effects were observed: (1) a decreased rate of cilia growth; (2) a lower cilia density that did not change over time; and (3) a wide distribution of cilia lengths that increased over time. These results confirm the importance of dynein-2 in regulating ciliary length in Tetrahymena. Cell Motil. Cytoskeleton, 2009. (c) 2009 Wiley-Liss, Inc.

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.

Dynein-2 affects the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena thermophila.

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

Defects in ciliary localization of Nek8 is associated with cystogenesis.

Mutations in the human NIMA (Never in Mitosis gene A)-related kinase 8 (Nek8) are associated with a rare form of the juvenile renal cystic disease, nephronophthisis type 9, and mutations in murine Nek8 cause renal cysts in jck mice. Cystogenesis involves dysfunctional ciliary signaling, and we have previously reported that Nek8 localizes to the primary cilium in mouse kidney epithelial cells. We now report that in developing mouse kidney, Nek8 is detected in the cilia of a subset of ureteric-bud-derived tubules at embryonic day (E)15.5. An increasing proportion of ureteric-bud-derived tubules express ciliary Nek8 until E18.5. Postnatal day 1 and 7 Nek8 is observed with equal frequency in both ureteric-bud and non-ureteric-bud-derived tubules. To investigate the cell biological consequences of kinase-deficient and jck mutant forms of Nek8, we transiently expressed green fluorescent protein (GFP)-tagged constructs in vitro. Mutations in the kinase and C-terminal domains of Nek8 adversely affected ciliary targeting but did not affect ciliogenesis or ciliary length. Consistent with these in vitro observations, kidneys from homozygous jck mice revealed reduced ciliary expression of Nek8 compared with kidneys from heterozygous (unaffected) mice. These data indicate that the ciliary localization of Nek8 in a subset of ureteric-bud-derived kidney tubules is essential for maintaining the integrity of those tubules in the mammalian kidney.

IP 6 Got Rhythm?

Motile cilia are plasma membrane projections that contain a central core of microtubules whose movement relative to each other contributes to ciliary beating. The left-right body patterning of developing embryos is thought to result from cilia-dependent differential distribution of morphogens. Knockout of inositol 1,3,4,5,6-pentakisphosphate (IP 5 ) 2-kinase (Ipk1), the enzyme that synthesizes IP 6 , in zebrafish embryos results in defective left-right positioning of organs, although the mechanism involved is unknown. Sarmah et al . extended this work and knocked down expression of ipk1 in zebrafish by injecting embryos with an antisense morpholino oligonucleotide (MO) targeted to ipk1 mRNA ( ipk1 MO1 embryos). The Kupffer’s vesicle (KV) is the organ that initiates left-right organ placement in the zebrafish. The authors found that whereas KV cilia from wild-type embryos beat with a regular frequency in a counterclockwise direction, those from ipk1 MO1 embryos vibrated without a defined direction of motion. Furthermore, immunocytochemical analyses revealed that cilia in ipk1 MO1 embryos were shorter than those in wild-type embryos. Although the ectopic expression in ipk1 MO1 embryos of Ipk1 resulted in the rescue of ciliary length and motion, that of a catalytically inactive mutant of Ipk1 did not. Fluorescence microscopy showed that Ipk1 was localized to centrosomes in the basal bodies of cilia in wild-type embryos. Transmission electron microscopy demonstrated that the organization of microtubules in the cilia from ipk1 MO1 embryos was similar to that in wild-type embryos. However, the anterograde microtubule-dependent movement of melanosomes (pigmented vesicles), which is mediated by kinesin proteins, was much slower in ipk1 MO1 cilia than in wild-type cilia. Together these data implicate Ipk1, and potentially its product IP 6 , in the maintenance of cilia length and the regulation of ciliary motion. B. Sarmah, V. P. Winfrey, G. E. Olson, B. Appel, S. R. Wente, A role for the inositol kinase Ipk1 in ciliary beating and length maintenance. Proc. Natl. Acad. Sci. U.S.A. 104 , 19843-19848 (2007). [Abstract] [Full Text]

A role for the inositol kinase Ipk1 in ciliary beating and length maintenance

Cilia project from cells as membranous extensions, with microtubule structural cores assembling from basal bodies by intraflagellar transport (IFT). Here, we report a ciliary role for the inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1) that generates inositol hexakisphosphate. In zebrafish embryos, reducing Ipk1 levels inhibited ciliary beating in Kupffer's vesicle and decreased ciliary length in the spinal canal, pronephric ducts, and Kupffer's vesicle. Electron microscopy showed that ciliary axonemal structures were not grossly altered. However, coincident knockdown of Ipk1 and IFT88 or IFT57 had synergistic perturbations. With GFP-Ipk1 enriched in centrosomes and basal bodies, we propose that Ipk1 plays a previously uncharacterized role in ciliary function.