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

1985 ◽  
Vol 101 (1) ◽  
pp. 130-140 ◽  
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.

Imaging of plasmacytoid dendritic cell interactions with T cells

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 75-84 ◽  
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.

scholarly journals Single-Particle Tracking Porcine Epidemic Diarrhea Virus Moving along Microtubules in Living Cells

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 124
Author(s):  
Yangyang Li ◽  
Wei Hou ◽  
Jian Wang ◽  
Fei Liu
Keyword(s):  
Particle Tracking ◽  
Single Particle ◽  
Porcine Epidemic Diarrhea Virus ◽  
Living Cells ◽  
Single Particle Tracking ◽  
Microtubule Organizing Center ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Organizing Center ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused severe damage to the swine industry. Although viruses are believed to hijack the microtubule-based transport system, the exact manner of PEDV moving along microtubules has not been fully characterized. In this study, PEDV was labeled with quantum dots which have great brightness and photostability. By using quantum dot-labeled PEDV and single-particle tracking, we were able to systematically dissect the dynamic behaviors of PEDV moving along the microtubules in living cells. We found that PEDVs maintained a restricted motion mode with a relatively stable speed in the cell membrane region while displaying a slow–fast–slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule-organizing center. The return movements of small amounts of PEDVs were also observed in living cells. Collectively, our work is crucial for understanding the movement of PEDV in living cells; the proposed work also provides important references for further analysis and studies of the infection mechanism of PEDV.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

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

1981 ◽  
Vol 91 (2) ◽  
pp. 589-594 ◽  
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.

scholarly journals Translocation and clustering of endosomes and lysosomes depends on microtubules.

1987 ◽  
Vol 105 (3) ◽  
pp. 1253-1265 ◽  
Author(s):  
R Matteoni ◽  
T E Kreis
Keyword(s):  
Rat Kidney ◽  
Living Cells ◽  
Cytochalasin D ◽  
Microtubule Organizing Center ◽  
Enhanced Fluorescence ◽  
Organelle Movement ◽  
Organizing Center ◽  
Normal Rat ◽  
Immunofluorescence Labeling

Indirect immunofluorescence labeling of normal rat kidney (NRK) cells with antibodies recognizing a lysosomal glycoprotein (LGP 120; Lewis, V., S.A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100:1839-1847) reveals that lysosomes accumulate in the region around the microtubule-organizing center (MTOC). This clustering of lysosomes depends on microtubules. When the interphase microtubules are depolymerized by treatment of the cells with nocodazole or during mitosis, the lysosomes disperse throughout the cytoplasm. Lysosomes recluster rapidly (within 30-60 min) in the region of the centrosomes either upon removal of the drug, or, in telophase, when repolymerization of interphase microtubules has occurred. During this translocation process the lysosomes can be found aligned along centrosomal microtubules. Endosomes and lysosomes can be visualized by incubating living cells with acridine orange. We have analyzed the movement of these labeled endocytic organelles in vivo by video-enhanced fluorescence microscopy. Translocation of endosomes and lysosomes occurs along linear tracks (up to 10 microns long) by discontinuous saltations (with velocities of up to 2.5 microns/s). Organelles move bidirectionally with respect to the MTOC. This movement ceases when microtubules are depolymerized by treatment of the cells with nocodazole. After nocodazole washout and microtubule repolymerization, the translocation and reclustering of fluorescent organelles predominantly occurs in a unidirectional manner towards the area of the MTOC. Organelle movement remains unaffected when cells are treated with cytochalasin D, or when the collapse of intermediate filaments is induced by microinjected monoclonal antivimentin antibodies. It can be concluded that translocation of endosomes and lysosomes occurs along microtubules and is independent of the intermediate filament and microfilament networks.

scholarly journals Visualization of the intracellular behavior of HIV in living cells

2002 ◽  
Vol 159 (3) ◽  
pp. 441-452 ◽  
Author(s):  
David McDonald ◽  
Marie A. Vodicka ◽  
Ginger Lucero ◽  
Tatyana M. Svitkina ◽  
Gary G. Borisy ◽  
...  
Keyword(s):  
Life Cycle ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Organizing Center ◽  
Microtubule Network ◽  
Organizing Center ◽  
Infected Cells ◽  
Resolution Imaging ◽  
Transcription Complexes ◽  
Hiv 1

To track the behavior of human immunodeficiency virus (HIV)-1 in the cytoplasm of infected cells, we have tagged virions by incorporation of HIV Vpr fused to the GFP. Observation of the GFP-labeled particles in living cells revealed that they moved in curvilinear paths in the cytoplasm and accumulated in the perinuclear region, often near the microtubule-organizing center. Further studies show that HIV uses cytoplasmic dynein and the microtubule network to migrate toward the nucleus. By combining GFP fused to the NH2 terminus of HIV-1 Vpr tagging with other labeling techniques, it was possible to determine the state of progression of individual particles through the viral life cycle. Correlation of immunofluorescent and electron micrographs allowed high resolution imaging of microtubule-associated structures that are proposed to be reverse transcription complexes. Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle.

scholarly journals Inflammasome activation in neutrophils of patients with severe COVID-19

2022 ◽  
Author(s):  
Karen Aymonnier ◽  
Julie Ng ◽  
Laura E Fredenburgh ◽  
Katherin Zambrano-Vera ◽  
Patrick Münzer ◽  
...  
Keyword(s):  
Time Lapse ◽  
Sterile Inflammation ◽  
Inflammasome Activation ◽  
Microtubule Organizing Center ◽  
Reporter Mice ◽  
Organizing Center ◽  
Monocytes And Macrophages ◽  
Histone Citrullination ◽  
Net Release

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.

scholarly journals Maturing Autophagosomes are Transported Towards the Cell Periphery

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.

Three-Dimensional reconstruction of isolated centrosomes by automated electron tomography

1994 ◽  
Vol 52 ◽  
pp. 310-311
Author(s):  
M.B. Braunfeld ◽  
M. Moritz ◽  
B.M. Alberts ◽  
J.W. Sedat ◽  
D.A. Agard
Keyword(s):  
Electron Tomography ◽  
Three Dimensional ◽  
Vital Role ◽  
Complex Nature ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Nucleation Sites ◽  
Dimensional Reconstruction ◽  
Organizing Center ◽  
Resolution Model

In animal cells, the centrosome functions as the primary microtubule organizing center (MTOC). As such the centrosome plays a vital role in determining a cell's shape, migration, and perhaps most importantly, its division. Despite the obvious importance of this organelle little is known about centrosomal regulation, duplication, or how it nucleates microtubules. Furthermore, no high resolution model for centrosomal structure exists.We have used automated electron tomography, and reconstruction techniques in an attempt to better understand the complex nature of the centrosome. Additionally we hope to identify nucleation sites for microtubule growth.Centrosomes were isolated from early Drosophila embryos. Briefly, after large organelles and debris from homogenized embryos were pelleted, the resulting supernatant was separated on a sucrose velocity gradient. Fractions were collected and assayed for centrosome-mediated microtubule -nucleating activity by incubating with fluorescently-labeled tubulin subunits. The resulting microtubule asters were then spun onto coverslips and viewed by fluorescence microscopy.

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