Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

We show here that human embryonic stem (ES) and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide—in particular, saturated, long-chain C_16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C_24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C_24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.     

Effects of Inhibition of Serine Palmitoyltransferase (SPT) and Sphingosine Kinase 1 (SphK1) on Palmitate Induced Insulin Resistance in L6 Myotubes

Background

The objective of this study was to examine the effects of short (2 h) and prolonged (18 h) inhibition of serine palmitoyltransferase (SPT) and sphingosine kinase 1 (SphK1) on palmitate (PA) induced insulin resistance in L6 myotubes.

Methods

L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor) or PA and Ski II (SphK1inhibitor) for different time periods (2 h and 18 h). Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA), ceramide (CER), sphingosine (SFO), sphingosine-1-phosphate (S1P)] were estimated by HPLC.

Results

Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio) in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes.

Conclusion

Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor). Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.     

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas .     

Modulating serine palmitoyl transferase (SPT) expression and activity unveils a crucial role in lipid-induced insulin resistance in rat skeletal muscle cells

Saturated fatty acids, such as palmitate, promote accumulation of ceramide, which impairs activation and signalling of PKB (protein kinase B; also known as Akt) to important end points such as glucose transport. SPT (serine palmitoyl transferase) is a key enzyme regulating ceramide synthesis from palmitate and represents a potential molecular target in curbing lipid-induced insulin resistance. In the present study we explore the effects of palmitate upon insulin action in L6 muscle cells in which SPT expression/activity has been decreased by shRNA (small-hairpin RNA) or sustained incubation with myriocin, an SPT inhibitor. Incubation of L6 myotubes with palmitate (for 16 h) increases intramyocellular ceramide and reduces insulin-stimulated PKB activation and glucose uptake. PKB inhibition was not associated with impaired IRS (insulin receptor substrate) signalling and was ameliorated by short-term treatment with myriocin. Silencing SPT expression (approximately 90%) by shRNA or chronic cell incubation with myriocin (for 7 days) markedly suppressed SPT activity and palmitate-driven ceramide synthesis; however, challenging these muscle cells with palmitate still inhibited the hormonal activation of PKB. This inhibition was associated with reduced IRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arises from diverting palmitate towards greater DAG (diacylglycerol) synthesis, which elevates IRS1 serine phosphorylation via activation of DAG-sensitive PKCs (protein kinase Cs). Treatment of SPT-shRNA cells or those treated chronically with myriocin with PKC inhibitors antagonized palmitate-induced loss in insulin signalling. The findings of the present study indicate that SPT plays a crucial role in desensitizing muscle cells to insulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-induced insulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAG synthesis, which impacts negatively upon IRS1-directed insulin signalling.     

Peroxiredoxin 1 is involved in disassembly of flagella and cilia

Cilia/flagella are evolutionarily conserved cellular organelles. In this study, we demonstrated that Dunaliella salina Peroxiredoxin 1 (DsPrdx1) localized to the flagella and basal bodies, and was involved in flagellar disassembly. The link between DsPrdx1 and flagella of Dunaliella salina (D. salina) encouraged us to explore the function of its human homologue, Homo sapiens Peroxiredoxin 1 (HsPrdx1) in development and physiology. Our results showed that HsPrdx1 was overexpressed, and cilia were lost in esophageal squamous cell carcinoma (ESCC) cells compared with the non-cancerous esophageal epithelial cells Het-1A. Furthermore, when HsPrdx1 was knocked down by short hairpin RNA (shRNA) lentivirus in ESCC cells, the phenotype of cilia lost can be reversed, and the expression levels of tumor suppressor genes LKB1 and p-AMPK were increased, and the activity of the oncogene Aurora A was inhibited compared with those in cells transfected with scrambe-shRNA lentivirus. These findings firstly showed that Prdx1 is involved in disassembly of flagella and cilia, and suggested that the abnormal expression of the cilia-related gene including Prdx1 may affect both ciliogenesis and cancernogenesis.     

The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization

Directional flow of the cerebrospinal fluid requires coordinated movement of the motile cilia of the ependymal epithelium that lines the cerebral ventricles. Here we report that mice lacking the Na⁺/H⁺ Exchanger Regulatory Factor 1 (NHERF1/Slc9a3r1, also known as EBP50) develop profound communicating hydrocephalus associated with fewer and disorganized ependymal cilia. Knockdown of NHERF1/slc9a3r1 in zebrafish embryos also causes severe hydrocephalus of the hindbrain and impaired ciliogenesis in the otic vesicle. Ultrastructural analysis did not reveal defects in the shape or organization of individual cilia. Similar phenotypes have been described in animals with deficiencies in Wnt signaling and the Planar Cell Polarity (PCP) pathway. We show that NHERF1 binds the PCP core genes Frizzled (Fzd) and Vangl. We further show that NHERF1 assembles a ternary complex with Fzd4 and Vangl2 and promotes translocation of Vangl2 to the plasma membrane, in particular to the apical surface of ependymal cells. Taken together, these results strongly support an important role for NHERF1 in the regulation of PCP signaling and the development of functional motile cilia.     

Regulation of primary cilia formation by ceramide

The primary cilium is an important sensory organelle, the regulation of which is not fully understood. We found that in polarized Madin-Darby Canine Kidney cells, the sphingolipid ceramide is specifically distributed to a cis-Golgi compartment at the base of the primary cilium. This compartment immunostained for the centrosome marker γ-tubulin, the Rho type GTPase cell division cycle 42 (Cdc42), and atypical protein kinase Cζ/λ (aPKC), a kinase activated by ceramide and associated with a polarity protein complex consisting of partitioning defective (Par)6 and Cdc42. Inhibition of ceramide biosynthesis with Fumonisin B1 prevented codistribution of aPKC and Cdc42 in the centrosomal/pericentriolar compartment and severely impaired ciliogenesis. Cilium formation and codistribution of aPKC and Cdc42 were restored by incubation with N-acetyl or N-palmitoyl sphingosine (C2 or C16 ceramide), or the ceramide analog N-oleoyl serinol (S18). Cilium formation was also restored by the glycogen synthase kinase-3β (GSK-3β) inhibitor indirubin-3-monoxime, suggesting that regulation of ciliogenesis depends on the inhibition of GSK-3β by ceramide-activated aPKC. Consistently, inhibition of aPKC with a pseudosubstrate inhibitor prevented restoration of ciliogenesis by C2 ceramide or S18. Our data show for the first time that ceramide is required for primary cilium formation.—Wang, G., K. Krishnamurthy, and E. Bieberich. Regulation of primary cilia formation by ceramide.     

High-Throughput Phenotyping of Chlamydomonas Swimming Mutants Based on Nanoscale Video Analysis

Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.     

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

Background

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Methods

The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.

Results

We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.

Conclusions

The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.

General significance

Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.     

Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity,Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.     

Centrin2 regulates CP110 removal in primary cilium formation

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.     

Stress-Induced Sphingolipid Signaling: Role of Type-2 Neutral Sphingomyelinase in Murine Cell Apoptosis and Proliferation

Background

Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg²⁺-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations.

Methodology and Principal Findings

For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation.

Conclusion and Significance

nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.     

Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1–depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.     

Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.     

The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes δ-Tubulin, a New Member of the Tubulin Superfamily

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.     

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.     

Neutral sphingomyelinase 2 deficiency is associated with lung anomalies similar to emphysema

Neutral sphingomyelinase 2 (nSMase2) upregulation was recently demonstrated to serve as a molecular link between smoke inhalation and emphysematous changes in lungs. Here we report that nSMase2 deficit impairs lung development in mice. We have shown previously that fragilitas ossium (fro) mice carry a mutation in the Smpd3 gene, rendering nSMase2 catalytically inactive. Analysis of lung phenotype revealed that fro mice have abnormally enlarged alveoli and increased compliance of the respiratory system, similar to morphological and functional manifestations of emphysema. Analysis of sphingolipid content in fro lungs revealed a decreased level of C14:0 ceramide but no significant alterations in the levels of sphingosine or sphingosine-1-phosphate. Altogether, our data suggest that nSMase2 activity and ceramide level are critical for lung development and function. Based on our data, ceramide can no longer be viewed as a lipid solely detrimental to lung function.     

ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.