A contractile activity that closes phagosomes in macrophages

1999 ◽  
Vol 112 (3) ◽  
pp. 307-316 ◽  
Author(s):  
J.A. Swanson ◽  
M.T. Johnson ◽  
K. Beningo ◽  
P. Post ◽  
M. Mooseker ◽  
...  
Keyword(s):  
Immunofluorescence Microscopy ◽  
Contractile Activity ◽  
Particle Surface ◽  
Microscopic Analysis ◽  
Time Lapse ◽  
Purse String ◽  
Mouse Macrophages ◽  
Butanedione Monoxime ◽  
Phosphoinositide 3 Kinase ◽  
Uncompetitive Inhibitor

Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.

scholarly journals Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes.

1994 ◽  
Vol 179 (2) ◽  
pp. 601-608 ◽  
Author(s):  
C M Alpuche-Aranda ◽  
E L Racoosin ◽  
J A Swanson ◽  
S I Miller
Keyword(s):  
Bone Marrow ◽  
Plasma Membrane ◽  
Time Lapse ◽  
Membrane Ruffling ◽  
Mouse Macrophages ◽  
Constitutive Mutation ◽  
Microscopic Studies ◽  
Specific Igg ◽  
Salmonella Survival ◽  
Time Lapse Video

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosomes, consistent with that expected for conventional receptor-mediated phagocytosis. These results suggest that formation of SP contributes to Salmonella survival and virulence.

scholarly journals Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

10.3791/57566 ◽  
2018 ◽  
Author(s):  
Markus Horsthemke ◽  
Janine Wilden ◽  
Anne C. Bachg ◽  
Peter J. Hanley
Keyword(s):  
3D Imaging ◽  
Time Lapse ◽  
Mouse Macrophages

Time-lapse microscopic analysis to verify how blastomere biopsy for PGD affects the dynamics of embryonic development

2014 ◽  
Vol 102 (3) ◽  
pp. e19-e20 ◽  
Author(s):  
D. Ben-Yosef ◽  
L. Bar-El ◽  
T. Shwartz ◽  
T. Cohen ◽  
A. Carmon ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Microscopic Analysis ◽  
Time Lapse ◽  
Blastomere Biopsy

scholarly journals Caveolae and Endoplasmic Reticulum: Immunofluorescence Microscopy and Time-Lapse Analysis.

1997 ◽  
Vol 30 (5/6) ◽  
pp. 593-599 ◽  
Author(s):  
Hiroshi Kogo ◽  
Mariko Shioya ◽  
Yukiko Takahashi ◽  
Toyoshi Fujimoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunofluorescence Microscopy ◽  
Time Lapse

scholarly journals Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia.

1979 ◽  
Vol 83 (2) ◽  
pp. 320-337 ◽  
Author(s):  
E Wang ◽  
R K Cross ◽  
P W Choppin
Keyword(s):  
Protein Synthesis ◽  
Cell Fusion ◽  
Structural Integrity ◽  
Immunofluorescence Microscopy ◽  
Time Lapse ◽  
Nuclear Migration ◽  
High Concentration ◽  
Nuclear Movement ◽  
Filament Bundles ◽  
10 Nm Filaments

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501-520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.

The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1653-1653 ◽  
Author(s):  
Joseph E. Italiano ◽  
Jennifer L. Richardson ◽  
Harald Schulze ◽  
Ksenija Drabek ◽  
Chloe Bulinski ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immunofluorescence Microscopy ◽  
Blood Platelet ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Cell Periphery ◽  
Structural Studies ◽  
Time Lapse Microscopy ◽  
Marginal Band ◽  
Tyrosinated Tubulin

Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.

scholarly journals Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7

Blood ◽  
2012 ◽  
Vol 120 (1) ◽  
pp. 199-206 ◽  
Author(s):  
Christian G. Peters ◽  
Alan D. Michelson ◽  
Robert Flaumenhaft
Keyword(s):  
Immunofluorescence Microscopy ◽  
Time Lapse ◽  
Granule Exocytosis ◽  
Time Lapse Microscopy ◽  
Gray Platelet Syndrome ◽  
Platelet Spreading ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Discrete Functions ◽  
Single Granule

Abstract There has been recent controversy as to whether platelet α-granules represent a single granule population or are composed of different subpopulations that serve discrete functions. To address this question, we evaluated the localization of vesicle-associated membrane proteins (VAMPs) in spread platelets to determine whether platelets actively sort a specific subpopulation of α-granules to the periphery during spreading. Immunofluorescence microscopy demonstrated that granules expressing VAMP-3 and VAMP-8 localized to the central granulomere of spread platelets along with the granule cargos von Willebrand factor and serotonin. In contrast, α-granules expressing VAMP-7 translocated to the periphery of spread platelets along with the granule cargos TIMP2 and VEFG. Time-lapse microscopy demonstrated that α-granules expressing VAMP-7 actively moved from the granulomere to the periphery during spreading. Platelets from a patient with gray platelet syndrome lacked α-granules and demonstrated only minimal spreading. Similarly, spreading was impaired in platelets obtained from Unc13dJinx mice, which are deficient in Munc13-4 and have an exocytosis defect. These studies identify a new α-granule subtype expressing VAMP-7 that moves to the periphery during spreading, supporting the premise that α-granules are heterogeneous and demonstrating that granule exocytosis is required for platelet spreading.

scholarly journals Myxococcus xanthus Displays Frz-Dependent Chemokinetic Behavior during Vegetative Swarming

1998 ◽  
Vol 180 (2) ◽  
pp. 440-443 ◽  
Author(s):  
Mandy J. Ward ◽  
Kenny C. Mok ◽  
David R. Zusman
Keyword(s):  
Signal Transduction ◽  
Life Cycle ◽  
Cell Density ◽  
Myxococcus Xanthus ◽  
Microscopic Analysis ◽  
Time Lapse ◽  
Complex Life Cycle ◽  
Signal Transduction System ◽  
Nutrient Levels ◽  
Time Lapse Video

ABSTRACT Myxococcus xanthus has been shown to utilize both directed (tactic) and undirected (kinetic) movements during different stages of its complex life cycle. We have used time-lapse video microscopic analysis to separate tactic and kinetic behaviors associated specifically with vegetatively swarming cells. Isolated individual cells separated by a thin agar barrier from mature swarms showed significant increases in gliding velocity compared to that of similar cells some distance from the swarm. This orthokinetic behavior was independent of the frequency of reversals of gliding direction (klinokinesis) but did require both the Frz signal transduction system and S-motility. We propose that M. xanthus uses Frz-dependent, auto-orthokinetic behavior to facilitate the dispersal of cells under conditions where both cell density and nutrient levels are high.

scholarly journals Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis

2016 ◽  
Vol 33 (11) ◽  
pp. 1449-1457 ◽  
Author(s):  
Liron Bar-El ◽  
Yael Kalma ◽  
Mira Malcov ◽  
Tamar Schwartz ◽  
Shaul Raviv ◽  
...  
Keyword(s):  
Microscopic Analysis ◽  
Time Lapse ◽  
Blastomere Biopsy
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