Inflammasome activation in neutrophils of patients with severe COVID-19

Infection by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-COV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation, (i.e. ASC speck assembly), and timing relative to NETosis in stimulated neutrophils by real time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that approximately 2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in LPS-stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center, long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

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Imaging of plasmacytoid dendritic cell interactions with T cells

Blood ◽

10.1182/blood-2008-02-139865 ◽

2009 ◽

Vol 113 (1) ◽

pp. 75-84 ◽

Cited By ~ 33

Author(s):

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

María López-Bravo ◽

Noa B. Martín-Cofreces ◽

Alix Scholer ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Plasmacytoid Dendritic Cell ◽

Time Lapse ◽

Cell Interactions ◽

Type I ◽

Microtubule Organizing Center ◽

Immune Synapse ◽

Organizing Center

Abstract Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4+ T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-θ, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC–T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

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The role of the ameiotic1 gene in the initiation of meiosis and in subsequent meiotic events in maize.

Genetics ◽

10.1093/genetics/135.4.1151 ◽

1993 ◽

Vol 135 (4) ◽

pp. 1151-1166 ◽

Cited By ~ 3

Author(s):

I Golubovskaya ◽

Z K Grebennikova ◽

N A Avalkina ◽

W F Sheridan

Keyword(s):

Meiotic Division ◽

Higher Plants ◽

Gene Interaction ◽

Microtubule Organizing Center ◽

Spindle Fiber ◽

Chromosome Synapsis ◽

Organizing Center ◽

Relationship Of ◽

The Relationship

Abstract Understanding the initiation of meiosis and the relationship of this event with other key cytogenetic processes are major goals in studying the genetic control of meiosis in higher plants. Our genetic and structural analysis of two mutant alleles of the ameiotic1 gene (am1 and am1-praI) suggest that this locus plays an essential role in the initiation of meiosis in maize. The product of the ameiotic1 gene affects an earlier stage in the meiotic sequence than any other known gene in maize and is important for the irreversible commitment of cells to meiosis and for crucial events marking the passage from premeiotic interphase into prophase I including chromosome synapsis. It appears that the period of ameiotic1 gene function in meiosis at a minimum covers the interval from some point during premeiotic interphase until the early zygotene stage of meiosis. To study the interaction of genes in the progression of meiosis, several double meiotic mutants were constructed. In these double mutants (i) the ameiotic1 mutant allele was brought together with the meiotic mutation (afd1) responsible for the fixation of centromeres in meiosis; and with the mutant alleles of the three meiotic genes that control homologous chromosome segregation (dv1, ms43 and ms28), which impair microtubule organizing center organization, the orientation of the spindle fiber apparatus, and the depolymerization of spindle filaments after the first meiotic division, respectively; (ii) the afd1 mutation was combined with two mutations (dsy1 and as1) affecting homologous pairing; (iii) the ms43 mutation was combined with the as1, the ms28 and the dv1 mutations; and (iv) the ms28 mutation was combined with the dv1 mutation and the ms4 (polymitotic1) mutations. An analysis of gene interaction in the double mutants led us to conclude that the ameiotic1 gene is epistatic over the afd1, the dv1, the ms43 and the ms28 genes but the significance of this relationship requires further analysis. The afd gene appears to function from premeiotic interphase throughout the first meiotic division, but it is likely that its function begins after the start of the ameiotic1 gene expression. The afd1 gene is epistatic over the two synaptic mutations dsy1 and as1 and also over the dv1 mutation. The new ameiotic*-485 and leptotene arrest*-487 mutations isolated from an active Robertson's Mutator stocks take part in the control of the initiation of meiosis.

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Centriole and Golgi microtubule nucleation are dispensable for the migration of human neutrophil-like cells

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0060 ◽

2021 ◽

pp. mbc.E21-02-0060

Author(s):

Lucas C. Klemm ◽

Ryan A. Denu ◽

Laurel E. Hind ◽

Briana L. Rocha-Gregg ◽

Mark E. Burkard ◽

...

Keyword(s):

Cell Line ◽

Host Defense ◽

Human Neutrophil ◽

Microtubule Organizing Center ◽

Microtubule Nucleation ◽

Directed Migration ◽

Inhibitory Role ◽

Organizing Center ◽

Cytoskeletal Rearrangements

Neutrophils migrate in response to chemoattractants to mediate host defense. Chemoattractants drive rapid intracellular cytoskeletal rearrangements including the radiation of microtubules from the microtubule-organizing center (MTOC) towards the rear of polarized neutrophils. Microtubules regulate neutrophil polarity and motility, but little is known about the specific role of MTOCs. To characterize the role of MTOCs on neutrophil motility we depleted centrioles in a well-established neutrophil-like cell line. Surprisingly, both chemical and genetic centriole depletion increased neutrophil speed and chemotactic motility, suggesting an inhibitory role for centrioles during directed migration. We also found that depletion of both centrioles and GM130-mediated Golgi microtubule nucleation did not impair neutrophil directed migration. Taken together, our findings demonstrate an inhibitory role for centrioles and a resilient MTOC system in motile human neutrophil-like cells. [Media: see text] [Media: see text] [Media: see text]

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HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

Science ◽

10.1126/science.aas8995 ◽

2020 ◽

Vol 369 (6510) ◽

pp. eaas8995 ◽

Cited By ~ 2

Author(s):

Venkat Giri Magupalli ◽

Roberto Negro ◽

Yuzi Tian ◽

Arthur V. Hauenstein ◽

Giuseppe Di Caprio ◽

...

Keyword(s):

Immune Surveillance ◽

Inflammasome Activation ◽

Histone Deacetylase 6 ◽

Microtubule Organizing Center ◽

Pyrin Domain ◽

Microtubule Transport ◽

Organizing Center ◽

Aggresome Formation ◽

Inflammatory Caspases

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1β (IL-1β) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain–containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1β conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

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Distribution of microtubule organizing centers in migrating sheets of endothelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.91.2.589 ◽

1981 ◽

Vol 91 (2) ◽

pp. 589-594 ◽

Cited By ~ 159

Author(s):

A I Gotlieb ◽

L M May ◽

L Subrahmanyan ◽

V I Kalnins

Keyword(s):

Endothelial Cells ◽

Monolayer Culture ◽

Cell Movement ◽

Time Lapse ◽

Microtubule Organizing Center ◽

Organizing Center ◽

Direction Of Movement ◽

The Relationship ◽

Wounded Area

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.

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The Role of the Centrosome in Development and Progression of Breast Cancer

Microscopy and Microanalysis ◽

10.1017/s1431927600028981 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 582-583

Author(s):

W. Lingle ◽

J. Salisbury ◽

S. Barrett ◽

V. Negron ◽

C. Whitehead

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Microtubule Organizing Center ◽

Daughter Cells ◽

Spindle Poles ◽

Sister Chromatids ◽

Close Proximity ◽

Interphase Cells ◽

Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

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Cytoplasmic motions, rheology, and structure probed by a novel magnetic particle method.

The Journal of Cell Biology ◽

10.1083/jcb.101.1.130 ◽

1985 ◽

Vol 101 (1) ◽

pp. 130-140 ◽

Cited By ~ 111

Author(s):

P A Valberg ◽

D F Albertini

Keyword(s):

Magnetic Particles ◽

Particle Method ◽

Time Lapse ◽

Living Cells ◽

Cellular Motility ◽

Phagocytic Cells ◽

Microtubule Organizing Center ◽

Organizing Center ◽

Pulmonary Macrophages ◽

Field Decay

The motions of magnetic particles contained within organelles of living cells were followed by measuring magnetic fields generated by the particles. The alignment of particles was sensed magnetometrically and was manipulated by external fields, allowing non-invasive detection of particle motion as well as examination of cytoplasmic viscoelasticity. Motility and rheology data are presented for pulmonary macrophages isolated from lungs of hamsters 1 d after the animals had breathed airborne gamma-Fe2O3 particles. The magnetic directions of particles within phagosomes and secondary lysosomes were aligned, and the weak magnetic field produced by the particles was recorded. For dead cells, this remanent field was constant, but for viable macrophages, the remanent field decreased rapidly so that only 42% of its initial magnitude remained 5 min after alignment. A twisting field was applied perpendicular to the direction of alignment and the rate at which particles reoriented to this new direction was followed. The same twisting was repeated for particles suspended in a series of viscosity standards. Based on this approach, the low-shear apparent intracellular viscosity was estimated to be 1.2-2.7 X 10(3) Pa.s (1.2-2.7 X 10(4) poise). Time-lapse video microscopy confirmed the alignment of ingested particles upon magnetization and showed persistent cellular motility during randomization of alignment. Cytochalasin D and low temperature both reduced cytoplasmic activity and remanent-field decay, but affected rheology differently. Magnetic particles were observed in association with the microtubule organizing center by immunofluorescence microscopy; magnetization did not affect microtubule distribution. However, both vimentin intermediate filaments and f-actin reorganized after magnetization. These data demonstrate that magnetometry of isolated phagocytic cells can probe organelle movements, rheology, and physical properties of the cytoskeleton in living cells.

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Molecular Regulation of Cell Fate in Cerebral Ischemia: Role of the Inflammasome and Connected Pathways

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.159 ◽

2014 ◽

Vol 34 (12) ◽

pp. 1857-1867 ◽

Cited By ~ 28

Author(s):

George Trendelenburg

Keyword(s):

Cell Fate ◽

Current Knowledge ◽

Protein Complexes ◽

Sterile Inflammation ◽

Inflammasome Activation ◽

Stroke Incidence ◽

Multifactorial Diseases ◽

Relevant Role ◽

Metabolic Conditions

Analogous to Toll-like receptors, NOD-like receptors represent a class of pattern recognition receptors, which are cytosolic and constitute part of different inflammasomes. These large protein complexes are activated not only by different pathogens, but also by sterile inflammation or by specific metabolic conditions. Mutations can cause hereditary autoinflammatory systemic diseases, and inflammasome activation has been linked to many multifactorial diseases, such as diabetes or cardiovascular diseases. Increasing data also support an important role in different central nervous diseases such as stroke. Thus, the current knowledge of the functional role of this intracellular ‘master switch’ of inflammation is discussed with a focus on its role in ischemic stroke, neurodegeneration, and also with regard to the recent data which argues for a relevant role in other organs or biologic systems which influence stroke incidence or prognosis.

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Maturing Autophagosomes are Transported Towards the Cell Periphery

Cellular and Molecular Neurobiology ◽

10.1007/s10571-021-01116-0 ◽

2021 ◽

Author(s):

Anna Hilverling ◽

Eva M. Szegö ◽

Elisabeth Dinter ◽

Diana Cozma ◽

Theodora Saridaki ◽

...

Keyword(s):

Intracellular Trafficking ◽

Fluorescent Proteins ◽

Time Lapse ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Time Lapse Microscopy ◽

Organizing Center ◽

Acidic Vesicles ◽

Luminal Ph ◽

Autophagosome Maturation

AbstractAutophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem-fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. Graphic Abstract (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

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The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly

Eukaryotic Cell ◽

10.1128/ec.00083-15 ◽

2015 ◽

Vol 14 (9) ◽

pp. 898-907 ◽

Cited By ~ 19

Author(s):

Huiqing Hu ◽

Yi Liu ◽

Qing Zhou ◽

Sara Siegel ◽

Ziyin Li

Keyword(s):

Cell Cycle ◽

Rna Interference ◽

Trypanosoma Brucei ◽

Basal Body ◽

Essential Role ◽

Microtubule Organizing Center ◽

Content Type ◽

Evolutionarily Conserved ◽

Organizing Center

ABSTRACT The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei , an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei .

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