Dynamic Remodeling of Membrane Composition Drives Cell Cycle through Primary Cilia Excision.

The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration.

Various mammalian cells have autonomous cellular clocks that are produced by the transcriptional cycle of clock genes. Cellular clocks provide circadian rhythms for cellular functions via transcriptional and cytoskeletal regulation. The vast majority of mammalian cells possess a primary cilium, an organelle protruding from the cell surface. Here, we investigated the little-known relationship between circadian rhythm and primary cilia. The length and number of primary cilia showed circadian dynamics both in vitro and in vivo. The circadian rhythm of primary cilium length was abolished by SR9011 and Bmal1 knockout. A centrosomal protein, pericentrin, transiently accumulates in centriolar satellites, the base of primary cilia at the shortest cilia phase, and induces elongation of primary cilia at the longest cilia phase in the circadian rhythm of primary cilia. In addition, rhythmic cell migration during wound healing depends on the length of primary cilia and affects the rate of wound healing. Our findings demonstrate that the circadian dynamics of primary cilium length by clock genes control fibroblast migration and could provide new insights into chronobiology.

Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.

Cancer cell proliferation is affected by post-translational modifications of tubulin. Especially, overexpression or depletion of enzymes for modifications on the tubulin C-terminal region perturbs dynamic instability of the spindle body. Those modifications include processing of C-terminal amino acids of α-tubulin; detyrosination, and a removal of penultimate glutamic acid (Δ2). We previously found a further removal of the third last glutamic acid, which generates so-called Δ3-tubulin. The effects of Δ3-tubulin on spindle integrities and cell proliferation remain to be elucidated. In this study, we investigated the impacts of forced expression of Δ3-tubulin on the structure of spindle bodies and cell division in a pancreatic cancer cell line, PANC-1. Overexpression of HA-tagged Δ3-tubulin impaired the morphology and orientation of spindle bodies during cell division in PANC-1 cells. In particular, spindle bending was most significantly increased. Expression of EGFP-tagged Δ3-tubulin driven by the endogenous promoter of human TUBA1B also deformed and misoriented spindle bodies. Spindle bending and condensation defects were significantly observed by EGFP-Δ3-tubulin expression. Furthermore, EGFP-Δ3-tubulin expression increased the nuclear size in a dose-dependent manner of EGFP-Δ3-tubulin expression. The expression of EGFP-Δ3-tubulin tended to slow down cell proliferation. Taken together, our results demonstrate that Δ3-tubulin affects the spindle integrity and cell division.

Effects of long-acting muscarinic antagonists on promoting ciliary function in airway epithelium.

BACKGROUND: Mucociliary clearance (MCC) is an essential defense mechanism in airway epithelia for removing pathogens from the respiratory tract. Impaired ciliary functions and MCC have been demonstrated in asthma and chronic obstructive pulmonary disease (COPD). Long-acting muscarinic antagonists (LAMAs) are a major class of inhaled bronchodilators, which are used for treating asthma and COPD; however, the effects of LAMAs on ciliary function remain unclear. This study aimed to identify the effects of LAMAs on airway ciliary functions. METHODS: Wild-type BALB/c mice were treated with daily intranasal administrations of glycopyrronium for 7 days, and tracheal samples were collected. Cilia-driven flow and ciliary activity, including ciliary beat frequency (CBF), ciliary beating amplitude, effective stroke velocity, recovery stroke velocity and the ratio of effective stroke velocity to recovery stroke velocity, were analyzed by imaging techniques. Using in vitro murine models, tracheal tissues were transiently cultured in media with/without LAMAs, glycopyrronium or tiotropium, for 60 min. Cilia-driven flow and ciliary activity were then analyzed. Well-differentiated normal human bronchial epithelial (NHBE) cells were treated with glycopyrronium, tiotropium, or vehicle for 60 min, and CBF was evaluated. Several mechanistic analyses were performed. RESULTS: Intranasal glycopyrronium administration for 7 days significantly increased cilia-driven flow and ciliary activity in murine airway epithelium. In the murine tracheal organ culture models, treatment with glycopyrronium or tiotropium for 60 min significantly increased cilia-driven flow and ciliary activity in airway epithelium. Further, we confirmed that 60-min treatment with glycopyrronium or tiotropium directly increased CBF in well-differentiated NHBE cells. In the mechanistic analyses, neither treatment with glycopyrronium nor tiotropium affected intracellular calcium ion concentrations in well-differentiated NHBE cells. Glycopyrronium did not increase protein kinase A activity in well-differentiated NHBE cells. Moreover, glycopyrronium had no effect on extracellular adenosine triphosphate concentration. CONCLUSIONS: LAMAs exert a direct effect on airway epithelium to enhance ciliary function, which may improve impaired MCC in asthma and COPD. Further investigations are warranted to elucidate the underlying mechanisms of the effects of LAMAs on the promotion of airway ciliary function.

Knock-in of Labeled Proteins into 5'UTR Enables Highly Efficient Generation of Stable Cell Lines.

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biology technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5'UTR of the endogenous gene loci. With this 5'UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high efficiencies ranging from 50 to 80% in antibiotic selected cells. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5'UTR is an amenable site to generate cell lines for the stable expression of tagged proteins from endogenous loci in mammalian cells.Key words: CRISPR/Cas9, knock-in, primary cilium, UTR, tubulin.

Impaired mucociliary motility enhances antigen-specific nasal IgA immune responses to a cholera toxin-based nasal vaccine.

Nasal mucosal tissues are equipped with physical barriers, mucus and cilia, on their surface. The mucus layer captures inhaled materials, and the cilia remove the inhaled materials from the epithelial layer by asymmetrical beating. The effect of nasal physical barriers on the vaccine efficacy remains to be investigated. Tubulin tyrosine ligase-like family, member 1 (Ttll1) is an essential enzyme for appropriate movement of the cilia on respiratory epithelium, and its deficiency (Ttll1-KO) leads to mucus accumulation in the nasal cavity. Here, when mice were intra-nasally immunized with pneumococcal surface protein A (PspA, as vaccine antigen) together with cholera toxin (CT, as mucosal adjuvant), Ttll1-KO mice showed higher levels of PspA-specific IgA in the nasal wash and increased numbers of PspA-specific IgA-producing plasma cells in the nasal passages when compared with Ttll1 hetero (He) mice. Mucus removal by N-acetylcysteine did not affect the enhanced immune responses in Ttll1-KO mice versus Ttll1-He mice. Immunohistological and flow cytometry analyses revealed that retention time of PspA in the nasal cavity in Ttll1-KO mice was longer than that in Ttll1-He mice. Consistently, uptake of PspA by dendritic cells was higher in the nasopharynx-associated lymphoid tissue (NALT) of Ttll1-KO mice than that of Ttll1-He mice. These results indicate that the ciliary function of removing vaccine antigen from the NALT epithelial layer is a critical determinant of the efficacy of nasal vaccine.

Influenza A virus enhances ciliary activity and mucociliary clearance via TLR3 in airway epithelium.

BACKGROUND: Viral respiratory tract infections, such as influenza A virus (IAV), are common and life-threatening illnesses worldwide. The mechanisms by which viruses are removed from the respiratory tract are indispensable for airway host defense. Mucociliary clearance is an airway defense mechanism that removes pathogens from the respiratory tract. The coordination and modulation of the ciliary beating of airway epithelial cells play key roles in maintaining effective mucociliary clearance. However, the impact of respiratory virus infection on ciliary activity and mucociliary clearance remains unclear. METHODS: Tracheal samples were taken from wild-type (WT) and Toll-like receptor 3 (TLR3)-knockout (KO) mice. Transient organ culture of murine trachea was performed in the presence or absence of IAV, polyI:C, a synthetic TLR3 ligand, and/or reagents. Subsequently, cilia-driven flow and ciliary motility were analyzed. To evaluate cilia-driven flow, red fluorescent beads were loaded into culture media and movements of the beads onto the tracheal surface were observed using a fluorescence microscope. To evaluate ciliary motility, cilia tips were labeled with Indian ink diluted with culture medium. The motility of ink-labeled cilia tips was recorded by high-speed cameras. RESULTS: Short-term IAV infection significantly increased cilia-driven flow and ciliary beat frequency (CBF) compared with the control level in WT culture. Whereas IAV infection did not elicit any increases of cilia-driven flow and CBF in TLR3-KO culture, indicating that TLR3 was essential to elicit an increase of cilia-driven flow and CBF in response to IAV infection. TLR3 activation by polyI:C readily induced adenosine triphosphate (ATP) release from the trachea and increases of cilia-driven flow and CBF in WT culture, but not in TLR3-KO culture. Moreover, blockade of purinergic P2 receptors (P2Rs) signaling using P2R antagonist, suramin, suppressed polyI:C-mediated increases of cilia-driven flow and CBF, indicating that TLR3-mediated ciliary activation depended on released extracellular ATP and the autocrine ATP-P2R loop. CONCLUSIONS: IAV infection readily increases ciliary activity and cilia-driven flow via TLR3 activation in the airway epithelium, thereby hastening mucociliary clearance and "sweeping" viruses from the airway as an initial host defense response. Mechanically, extracellular ATP release in response to TLR3 activation promotes ciliary activity through autocrine ATP-P2R loop.

Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques.

OBJECTIVE: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe-/-) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe-/- mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. CONCLUSIONS: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.

Role of caveolin-1 in hepatocellular carcinoma arising from non-alcoholic fatty liver disease.

The molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver disease (NAFLD-HCC) are not well known. In this study, we investigated the mechanism by which NAFLD-HCC survives in a fat-rich environment. We found that caveolin (CAV)-1 was overexpressed in clinical specimens from NAFLD-HCC patients. HepG2, HLE, and HuH-7 HCC cell lines showed decreased proliferation in the presence of the saturated fatty acids palmitic acid and stearic acid, although only HLE cells expressed high levels of CAV-1. HLE cells treated with oleic acid (OA) showed robust proliferation, whereas CAV-null HepG2 cells showed reduced proliferation and increased apoptosis. CAV-1 knockdown in HLE cells attenuated the OA-induced increase in proliferation and enhanced apoptosis. Liquid chromatography-tandem mass spectrometry analysis revealed that the levels of OA-containing ceramide, a pro-apoptotic factor, were higher in HepG2 and CAV-1-deficient HLE cells than in HLE cells, suggesting that CAV-1 inhibits apoptosis by decreasing the level of OA-containing ceramide. These results indicate that CAV-1 is important for NAFLD-HCC survival in fatty acid-rich environments and is a potential therapeutic target.

Stearate-to-palmitate ratio modulates endoplasmic reticulum stress and cell apoptosis in non-B non-C hepatoma cells.

The increased prevalence of hepatocellular carcinoma (HCC) without viral infection, namely, NHCC, is a major public health issue worldwide. NHCC is frequently derived from non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis, which exhibit dysregulated fatty acid (FA) metabolism. This raises the possibility that NHCC evolves intracellular machineries to adapt to dysregulated FA metabolism. We herein aim to identify NHCC-specifically altered FA and key molecules to achieve the adaptation. To analyze FA, imaging mass spectrometry (IMS) was performed on 15 HCC specimens. The composition of saturated FA (SFA) in NHCC was altered from that in typical HCC. The stearate-to-palmitate ratio (SPR) was significantly increased in NHCC. Associated with the SPR increase, the ELOVL6 protein level was upregulated in NHCC. The knockdown of ELOVL6 reduced SPR, and enhanced endoplasmic reticulum stress, inducing apoptosis of Huh7 and HepG2 cells. In conclusion, NHCC appears to adapt to an FA-rich environment by modulating SPR through ELOVL6.

Ttll9-/- mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating.

Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9(-/-)) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9(-/-) sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9(-/-) spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after anti-hook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

Prognostic value of chromosomal translocations in small-bowel diffuse large B-cell lymphoma.

AIMS: The objective of this study was to investigate the incidence and clinical significance of lymphoma-associated chromosomal translocations, particularly those involving the immunoglobulin heavy chain gene (IGH) locus, in patients with small-bowel diffuse large B-cell lymphoma (DLBCL). METHODS AND RESULTS: Translocations involving IGH, bcl-6, MYC and bcl-2 were investigated with interphase fluorescence in-situ hybridization on paraffin-embedded tissues in 35 patients with primary small-bowel DLBCL, and the overall survival (OS) and progression-free survival (PFS) rates were evaluated with the Kaplan-Meier method. Translocations involving IGH, bcl-6, MYC and bcl-2 were detected in 23 (70%), 12 (36%), eight (24%) and six (18%) of 33 cases, respectively. The patients with IGH translocations showed less frequent relapse or progression of lymphoma (17%) than those without (60%, P = 0.034). Univariate analyses demonstrated that young age, a low international prognostic index, translocations involving IGH, extra copies of MALT1/bcl-2 and bcl-6 immunoexpression were significantly associated with better OS and PFS. Cox multivariate analysis revealed translocations involving IGH to constitute an independent prognostic factor for better PFS, but not better OS. CONCLUSIONS: Translocations involving IGH are frequent in cases of small-bowel DLBCL. These translocations may be predictive of a favourable clinical course.

Direct profiling of the phospholipid composition of adult Caenorhabditis elegans using whole-body imaging mass spectrometry.

A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.

Therapeutic strategy for Crohn's disease with a loss of response to infliximab: a single-center retrospective study.

BACKGROUND/AIMS: Infliximab (IFX) is an effective treatment for maintaining clinical remission in patients with initially moderate-to-severe Crohn's disease (CD). However, a certain number of patients become unresponsive to IFX, subsequently requiring intensified therapy. The aim of this study was to compare the short- and long-term therapeutic efficacy of intensified regimens in CD patients who fail to respond to IFX. METHODS: The clinical courses of 33 CD patients who failed to respond to treatment with IFX were investigated retrospectively. An intensified regimen involving doubling the dose of IFX was chosen in 13 patients (DD group) versus shortening the IFX interval in 13 patients (SI group) and switching to adalimumab (ADA) in 7 patients (SA group). RESULTS: The clinical response and rate of clinical remission at 4 weeks were 62 and 54% in the DD group, 77 and 62% in the SI group and 57 and 43% in the SA group, respectively (p = 0.59 for clinical response, p = 0.90 for clinical remission). The rate of sustained remission at 48 weeks was 44% in the DD group, 54% in the SI group and 33% in the SA group (p = 0.88). CONCLUSION: The short- and long-term efficacy of doubling the dose of IFX, shortening the interval of IFX or switching to ADA is similar for CD patients who no longer respond to IFX.

Cytosolic carboxypeptidase 5 removes α- and γ-linked glutamates from tubulin.

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and ß-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and ß-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of ß3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.

Axonal gradient of arachidonic acid-containing phosphatidylcholine and its dependence on actin dynamics.

Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated. In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report of the gradual distribution of AA-PC along the axon and its association with actin dynamics.

Cytosolic carboxypeptidase 1 is involved in processing α- and ß-tubulin.

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and ß-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and ß-tubulin. The hyperglutamylation of α- and ß-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and ß-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from ß- as well as α-tubulin in vitro and in vivo.

Lysophosphatidylcholine acyltransferase 1 altered phospholipid composition and regulated hepatoma progression.

BACKGROUND & AIMS: Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS: Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines. RESULTS: IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa. CONCLUSIONS: Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.

Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry.

Airway epithelial cilia protect the mammalian respiratory system from harmful inhaled materials by providing the force necessary for effective mucociliary clearance. Ciliary beating is asymmetric, composed of clearly distinguished effective and recovery strokes. Neither the importance of nor the essential components responsible for the beating asymmetry has been directly elucidated. We report here that the beating asymmetry is crucial for ciliary function and requires tubulin glutamylation, a unique posttranslational modification that is highly abundant in cilia. WT murine tracheal cilia have an axoneme-intrinsic structural curvature that points in the direction of effective strokes. The axonemal curvature was lost in tracheal cilia from mice with knockout of a tubulin glutamylation-performing enzyme, tubulin tyrosine ligase-like protein 1. Along with the loss of axonemal curvature, the axonemes and tracheal epithelial cilia from these knockout (KO) mice lost beating asymmetry. The loss of beating asymmetry resulted in a reduction of cilia-generated fluid flow in trachea from the KO mice. The KO mice displayed a significant accumulation of mucus in the nasal cavity, and also emitted frequent coughing- or sneezing-like noises. Thus, the beating asymmetry is important for airway ciliary function. Our findings provide evidence that tubulin glutamylation is essential for ciliary function through the regulation of beating asymmetry, and provides insight into the molecular basis underlying the beating asymmetry.