Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

scholarly journals C3 Promotes Clearance of Klebsiella pneumoniae by A549 Epithelial Cells

2004 ◽  
Vol 72 (3) ◽  
pp. 1767-1774 ◽  
Author(s):  
Beatriz de Astorza ◽  
Guadalupe Cortés ◽  
Catalina Crespí ◽  
Carles Saus ◽  
José María Rojo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Alveolar Epithelial Cells ◽  
Clinical Isolates ◽  
Complement Components ◽  
Innate Defense ◽  
Alveolar Epithelial

ABSTRACT The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals Morphogenic Effects of Ezrin Require a Phosphorylation-Induced Transition from Oligomers to Monomers at the Plasma Membrane

2000 ◽  
Vol 150 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Wild Type ◽  
Erm Proteins ◽  
Threonine Residue ◽  
Phosphorylation Event ◽  
Membrane Bound

ERM (ezrin, radixin, moesin) proteins act as linkers between the plasma membrane and the actin cytoskeleton. An interaction between their NH2- and COOH-terminal domains occurs intramolecularly in closed monomers and intermolecularly in head-to-tail oligomers. In vitro, phosphorylation of a conserved threonine residue (T567 in ezrin) in the COOH-terminal domain of ERM proteins disrupts this interaction. Here, we have analyzed the role of this phosphorylation event in vivo, by deriving stable clones producing wild-type, T567A, and T567D ezrin from LLC-PK1 epithelial cells. We found that T567A ezrin was poorly associated with the cytoskeleton, but was able to form oligomers. In contrast, T567D ezrin was associated with the cytoskeleton, but its distribution was shifted from oligomers to monomers at the membrane. Moreover, production of T567D ezrin induced the formation of lamellipodia, membrane ruffles, and tufts of microvilli. Both T567A and T567D ezrin affected the development of multicellular epithelial structures. Collectively, these results suggest that phosphorylation of ERM proteins on this conserved threonine regulates the transition from membrane-bound oligomers to active monomers, which induce and are part of actin-rich membrane projections.

scholarly journals Phosphorylation of the microtubule-severing AAA+ enzyme Katanin regulates C. elegans embryo development

2020 ◽  
Vol 219 (6) ◽  
Author(s):  
Nicolas Joly ◽  
Eva Beaumale ◽  
Lucie Van Hove ◽  
Lisa Martino ◽  
Lionel Pintard
Keyword(s):  
Mitotic Spindle ◽  
The Other ◽  
Meiotic Spindle ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Evolutionarily Conserved ◽  
Necessary And Sufficient ◽  
Fine Tune

The evolutionarily conserved microtubule (MT)-severing AAA-ATPase enzyme Katanin is emerging as a critical regulator of MT dynamics. In Caenorhabditis elegans, Katanin MT-severing activity is essential for meiotic spindle assembly but is toxic for the mitotic spindle. Here we analyzed Katanin dynamics in C. elegans and deciphered the role of Katanin phosphorylation in the regulation of its activity and stability. Katanin is abundant in oocytes, and its levels drop after meiosis, but unexpectedly, a significant fraction is present throughout embryogenesis, where it is dynamically recruited to the centrosomes and chromosomes during mitosis. We show that the minibrain kinase MBK-2, which is activated during meiosis, phosphorylates Katanin at multiple serines. We demonstrate unequivocally that Katanin phosphorylation at a single residue is necessary and sufficient to target Katanin for proteasomal degradation after meiosis, whereas phosphorylation at the other sites only inhibits Katanin ATPase activity stimulated by MTs. Our findings suggest that cycles of phosphorylation and dephosphorylation fine-tune Katanin level and activity to deliver the appropriate MT-severing activity during development.

scholarly journals Caenorhabditis elegans nuclear RNAi factor SET-32 deposits the transgenerational histone modification, H3K23me3

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lianna Schwartz-Orbach ◽  
Chenzhen Zhang ◽  
Simone Sidoli ◽  
Richa Amin ◽  
Diljeet Kaur ◽  
...  
Keyword(s):  
Chromatin Modification ◽  
Epigenetic Inheritance ◽  
Rnai Pathway ◽  
C Elegans ◽  
And Function ◽  
Nuclear Rnai ◽  
Factor Set

Nuclear RNAi provides a highly tractable system to study RNA-mediated chromatin changes and epigenetic inheritance. Recent studies have indicated that the regulation and function of nuclear RNAi-mediated heterochromatin are highly complex. Our knowledge of histone modifications and the corresponding histonemodifying enzymes involved in the system remains limited. In this study, we show that the heterochromatin mark, H3K23me3, is induced by nuclear RNAi at both exogenous and endogenous targets in C. elegans. In addition, dsRNA-induced H3K23me3 can persist for multiple generations after the dsRNA exposure has stopped. We demonstrate that the histone methyltransferase SET-32, methylates H3K23 in vitro. Both set-32 and the germline nuclear RNAi Argonaute, hrde-1, are required for nuclear RNAi-induced H3K23me3 in vivo. Our data poise H3K23me3 as an additional chromatin modification in the nuclear RNAi pathway and provides the field with a new target for uncovering the role of heterochromatin in transgenerational epigenetic silencing.

ID: 112: ROLE OF PHOSPHOLIPASE D IN IDIOPATHIC PULMONARY FIBROSIS

2016 ◽  
Vol 64 (4) ◽  
pp. 964.1-964
Author(s):  
V Suryadevara ◽  
T Royston ◽  
E Berdyshev ◽  
L Huang ◽  
V Natarajan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Phospholipase D ◽  
Human Lung ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a deadly interstitial disease that leads to scarring and fibrosis of the lung tissue. In pulmonary fibrosis, there is injury and denudation of the alveolar epithelium, which further leads to activation of fibroblasts which differentiate into myofibroblasts. This includes several mechanisms including epithelial to mesenchymal transition (EMT). In this study, we investigated the role of phospholipase D (PLD) in IPF and also its underlying mechanism like EMT and fibroblast proliferation and differentiation. An in vivo murine model of bleomycin-induced pulmonary fibrosis (PF) and in vitro models of murine alveolar type-II epithelial cells (MLE-12) and human lung fibroblasts were used. C57BL/6 and genetically engineered PLD2−/− mice were intratracheally challenged with bleomycin (1.5 U/kg animal) for 14 days and markers of inflammation, EMT and fibrosis were determined. MLE-12 cells were treated with specific PLD1 or PLD2 inhibitors prior to bleomycin (10 mU/ml) challenge, and the role of PLD in EMT and apoptosis of alveolar epithelial cells was studied. Human lung fibroblasts were serum-starved (3h), pretreated with PLD1 or PLD2 inhibitors, and the effect of TGF-β (5 ng/ml) on differentiation of lung fibroblast to myofibroblast was determined. Intra-tracheal instillation of bleomycin in the mice for 14 days leads to the progression of fibrosis in the lung. The lung tissues of the bleomycin treated mice were found to have increased PLD2 protein expression, myofibroblast markers like α-SMA, fibronectin, mesenchymal markers like vimentin, inflammatory cytokines and collagen. Genetic deletion of PLD2 in mice attenuated bleomycin-induced lung inflammation and pulmonary fibrosis. In vitro, MLE-12 cells pretreated with either PLD1 or PLD2 inhibitor did not show a profound reduction either in apoptosis or the expression of transcription factors such as SNAIL, and other markers of EMT. However, MLE-12 cells pretreated with both PLD1 (250 nM) and PLD2 (500 nM) inhibitors were resistant to bleomycin-induced apoptosis, and exhibited reduced expression of SNAIL and mesenchymal markers. On the contrary, human lung fibroblasts pretreated with PLD1 and PLD2 inhibitors showed increased fibroblast to myofibroblast differentiation mediated by TGF-β. The present study suggests a role for PLD2 in bleomycin-induced PF. In vitro, inhibition of both PLD1 and PLD2 was necessary to attenuate bleomycin-induced EMT in epithelial cells and TGF-β mediated differentiation of fibroblasts to myofibroblasts. The in vivo and in vitro results identify the mechanism by which PLD regualtes PF and suggest PLD as a potential therapeutic target in pulmonary fibrosis. This work was supported by National Institutes of Health grant P01 HL98050 to VN.

scholarly journals Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication

2006 ◽  
Vol 80 (17) ◽  
pp. 8566-8581 ◽  
Author(s):  
Xuehua Zhong ◽  
Neocles Leontis ◽  
Shuiming Qian ◽  
Asuka Itaya ◽  
Yijun Qi ◽  
...  
Keyword(s):  
Essential Role ◽  
Tertiary Structure ◽  
Matrix Analysis ◽  
Base Pairs ◽  
Rna Motifs ◽  
Viroid Infection ◽  
Functional Analyses

ABSTRACT RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.

scholarly journals Autoamplification of NFATc1 expression determines its essential role in bone homeostasis

2005 ◽  
Vol 202 (9) ◽  
pp. 1261-1269 ◽  
Author(s):  
Masataka Asagiri ◽  
Kojiro Sato ◽  
Takako Usami ◽  
Sae Ochi ◽  
Hiroshi Nishina ◽  
...  
Keyword(s):  
Essential Role ◽  
Osteoclast Differentiation ◽  
Ectopic Expression ◽  
Epigenetic Mechanism ◽  
Bone Homeostasis ◽  
Hematopoietic Stem ◽  
Vitro Effect

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1−/− hematopoietic stem cells to osteoclast-deficient Fos−/− mice, and by Fos−/− blastocyst complementation, thus avoiding the embryonic lethality of NFATc1−/− mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.

Dimethyl sulfoxide decreases interleukin-8-mediated neutrophil recruitment in the airways

1996 ◽  
Vol 271 (5) ◽  
pp. L838-L843 ◽  
Author(s):  
P. P. Massion ◽  
A. Linden ◽  
H. Inoue ◽  
M. Mathy ◽  
K. M. Grattan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Dimethyl Sulfoxide ◽  
Neutrophil Recruitment ◽  
Interleukin 8 ◽  
Bronchial Epithelial ◽  
Anti Inflammatory

In this study, we investigated the role of dimethyl sulfoxide (DMSO) in inhibiting interleukin-8 (IL-8)-mediated neutrophil recruitment induced by Pseudomonas aeruginosa (PA) bacterial supernatant. First, we tested whether DMSO could inhibit IL-8 production induced by PA in human bronchial epithelial (16-HBE) cells in vitro. In these cells, exposure to PA or H2O2 induced IL-8 production dose dependently, an effect that was inhibited by 1% DMSO at both the protein and RNA level. Second, we tested whether DMSO could block the recruitment of neutrophils induced by PA in a bypassed segment of dog trachea in vivo. PA supernatant was placed in the tracheal segment for 6 h in four dogs, and neutrophil recruitment and IL-8 concentrations were measured in the superfusate. DMSO prevented the recruitment of neutrophils and IL-8 production induced by PA time dependently. The results suggest that DMSO may play an anti-inflammatory role in the airway by inhibiting IL-8 production in epithelial cells.

scholarly journals Dual oxidase 1 promotes antiviral innate immunity

2021 ◽  
Vol 118 (26) ◽  
pp. e2017130118
Author(s):  
Demba Sarr ◽  
Aaron D. Gingerich ◽  
Nuha Milad Asthiwi ◽  
Faris Almutairi ◽  
Giuseppe A. Sautto ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Influenza Virus ◽  
Epithelial Cells ◽  
Host Cells ◽  
Dependent Manner ◽  
Dual Oxidase ◽  
Antiviral Innate Immunity

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN−) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1−/− mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1β, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN− is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN− diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN− in an H2O2-dependent manner in vitro. OSCN− does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN−, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Sign in / Sign up

Export Citation Format

Share Document