The NIMA-family kinase,Nek1 affects the stability of centrosomes and ciliogenesis

Background  

Mutations in Nek1 (NIMA-Related Kinase 1) are causal in the murine models of polycystic kidney disease kat and kat ^2J . The Neks are known as cell cycle kinases, but recent work in protists has revealed that in addition to roles in the regulation of cell cycle progression, some Neks also regulate cilia. In most cells, cilia are disassembled prior to mitosis and are regenerated after cytokinesis. We propose that Neks participate in the coordination of ciliogenesis with cell cycle progression. Mammalian Nek1 is a candidate for this activity because renal cysts form in response to dysfunctional ciliary signalling.     

The radial organization of neuronal primary cilia is acutely disrupted by seizure and ischemic brain injury

Background

Neuronal primary cilia are sensory organelles that are critically involved in the proper growth, development, and function of the central nervous system (CNS). Recent work also suggests that they signal in the context of CNS injury, and that abnormal ciliary signaling may be implicated in neurological diseases.

Methods

We quantified the distribution of neuronal primary cilia alignment throughout the normal adult mouse brain by immunohistochemical staining for the primary cilia marker adenylyl cyclase III (ACIII) and measuring the angles of primary cilia with respect to global and local coordinate planes. We then introduced two different models of acute brain insult—temporal lobe seizure and cerebral ischemia, and re-examined neuronal primary cilia distribution, as well as ciliary lengths and the proportion of neurons harboring cilia.

Results

Under basal conditions, cortical cilia align themselves radially with respect to the cortical surface, while cilia in the dentate gyrus align themselves radially with respect to the granule cell layer. Cilia of neurons in the striatum and thalamus, by contrast, exhibit a wide distribution of ciliary arrangements. In both cases of acute brain insult, primary cilia alignment was significantly disrupted in a region-specific manner, with areas affected by the insult preferentially disrupted. Further, the two models promoted differential effects on ciliary lengths, while only the ischemia model decreased the proportion of ciliated cells.

Conclusions

These findings provide evidence for the regional anatomical organization of neuronal primary cilia in the adult brain and suggest that various brain insults may disrupt this organization.

     

A novel interaction between kinase activities in regulation of cilia formation

Background

The primary cilium is an extension of the cell membrane that encloses a microtubule-based axoneme. Primary cilia are essential for transmission of environmental cues that determine cell fate. Disruption of primary cilia function is the molecular basis of numerous developmental disorders. Despite their biological importance, the mechanisms governing their assembly and disassembly are just beginning to be understood. Cilia growth and disassembly are essential events when cells exit and reenter into the cell cycle. The kinases never in mitosis-kinase 2 (Nek2) and Aurora A (AurA) act to depolymerize cilia when cells reenter the cell cycle from G₀.

Results

Coexpression of either kinase with its kinase dead companion [AurA with kinase dead Nek2 (Nek2 KD) or Nek2 with kinase dead AurA (AurA KD)] had different effects on cilia depending on whether cilia are growing or shortening. AurA and Nek2 are individually able to shorten cilia when cilia are growing but both are required when cilia are being absorbed. The depolymerizing activity of each kinase is increased when coexpressed with the kinase dead version of the other kinase but only when cilia are assembling. Additionally, the two kinases act additively when cilia are assembling but not disassembling. Inhibition of AurA increases cilia number while inhibition of Nek2 significantly stimulates cilia length. The complex functional relationship between the two kinases reflects their physical interaction. Further, we identify a role for a PP1 binding protein, PPP1R42, in inhibiting Nek2 and increasing ciliation of ARPE-19 cells.

Conclusion

We have uncovered a novel functional interaction between Nek2 and AurA that is dependent on the growth state of cilia. This differential interdependence reflects opposing regulation when cilia are growing or shortening. In addition to interaction between the kinases to regulate ciliation, the PP1 binding protein PPP1R42 directly inhibits Nek2 independent of PP1 indicating another level of regulation of this kinase. In summary, we demonstrate a complex interplay between Nek2 and AurA kinases in regulation of ciliation in ARPE-19 cells.

     

The hydrocephalus inducing gene product, Hydin, positions axonemal central pair microtubules

Background  

Impairment of cilia and flagella function underlies a growing number of human genetic diseases. Mutations in hydin in hy3 mice cause lethal communicating hydrocephalus with early onset. Hydin was recently identified as an axonemal protein; however, its function is as yet unknown.     

A role for p38 MAPK in the regulation of ciliary motion in a eukaryote

Background  

Motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. Although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. Here we present evidence for p38 mitogen-activated protein kinase (p38 MAPK) playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite Schistosoma mansoni.     

TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

Objective

Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.

Methods and Results

We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.

Conclusion

We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.     

Smoking Is Associated with Shortened Airway Cilia

Background

Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6–7 µm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers.

Methodology/Principal Findings

Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers.

Conclusions/Significance

Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.     

Spatial distribution of calcium-gated chloride channels in olfactory cilia

Background

In 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 Findings

To 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.

Conclusions

On 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.     

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos

Background  

The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

Cilia are required for asymmetric <Emphasis Type="Italic">nodal</Emphasis> induction in the sea urchin embryo

Background

Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.

Results

Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.

Conclusions

Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

     

Association of sperm protein 17 with A-kinase anchoring protein 3 in flagella

Background  

Sperm protein 17 (Sp17) is a three-domain protein that contains: 1) a highly conserved N-terminal domain that is 45% identical to the human type II alpha regulatory subunit (RII alpha) of protein kinase A (PKA); 2) a central sulphated carbohydrate-binding domain; and 3) a C-terminal Ca++/calmodulin (CaM) binding domain. Although Sp17 was originally discovered and characterized in spermatozoa, its mRNA has now been found in a variety of normal mouse and human tissues. However, Sp17 protein is found predominantly in spermatozoa, cilia and human neoplastic cell lines. This study demonstrates that Sp17 from spermatozoa binds A-kinase anchoring protein 3 (AKAP3), confirming the functionality of the N-terminal domain.     

Development of ciliary bands in larvae of the living isocrinid sea lily Metacrinus rotundus

Embryos and larvae of an isocrinid sea lily, Metacrinus rotundus, are described by scanning electron microscopy. Around hatching (35 h after fertilization), the outer surface of the gastrula becomes ubiquitously covered with short cilia. At 40 h, the hatched swimming embryo develops a cilia‐free zone of ectoderm on the ventral side. By 3 days, the very early dipleurula larva develops a cilia‐free zone ventrally, densely ciliated regions laterally, and a sparsely ciliated region dorsally. At this stage, the posterior and anterior ciliary bands first appear: the former runs along a low ridge separating the densely from the sparsely ciliated epidermal regions, while the latter is visible, at first discontinuously, along the boundary between the densely ciliated lateral regions and the cilia‐free ventral zone. In the late dipleurula larva (5 days after fertilization), the anterior and posterior loops of ciliary bands are well defined. The transition from the dipleurula to the semidoliolaria larva occurs at 6 days as the posterior loop becomes rearranged to form incompletely circumferential ciliary bands. The larva becomes competent to settle at this stage. The arrangement of the ciliary bands on the semidoliolaria is maintained during the second week of development, while the larva retains its competence to settle. The larval ciliary patterns described here are compared with those of stalkless crinoids and eleutherozoan echinoderms. The closest morphological similarities are between M. rotundus and the basal eleutherozoan class Asteroidea.     

The Leber Congenital Amaurosis Protein AIPL1 and EB Proteins Co-Localize at the Photoreceptor Cilium

Purpose

The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).

Methods

The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.

Results

Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.

Conclusions

AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.     

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Background  

The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.