scholarly journals Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

1996 ◽  
Vol 134 (2) ◽  
pp. 455-464 ◽  
Author(s):  
H J Matthies ◽  
H B McDonald ◽  
L S Goldstein ◽  
W E Theurkauf
Keyword(s):  
Laser Scanning ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Meiotic Spindle ◽  
Bipolar Spindle ◽  
Scanning Confocal Microscopy ◽  
Spindle Poles ◽  
Assembly Kinetics

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

scholarly journals Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

1992 ◽  
Vol 116 (5) ◽  
pp. 1167-1180 ◽  
Author(s):  
W E Theurkauf ◽  
R S Hawley
Keyword(s):  
Laser Scanning ◽  
Metaphase Plate ◽  
Spindle Assembly ◽  
Structural Similarity ◽  
Laser Scanning Confocal Microscopy ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
X Chromosomes ◽  
Scanning Confocal Microscopy ◽  
Spindle Position

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

scholarly journals Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

2001 ◽  
Vol 75 (4) ◽  
pp. 1941-1948 ◽  
Author(s):  
Patrice Dunoyer ◽  
Etienne Herzog ◽  
Odile Hemmer ◽  
Christophe Ritzenthaler ◽  
Christiane Fritsch
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Viral Rna ◽  
Epifluorescence Microscopy ◽  
Scanning Confocal Microscopy ◽  
Virus Rna ◽  
Green Fluorescent ◽  
Rna Accumulation

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

SINGLE CELL IMAGING OF BAX TRANSLOCATION DURING APOPTOSIS INDUCED BY PHOTOFRIN-PDT

2009 ◽  
Vol 02 (02) ◽  
pp. 209-214
Author(s):  
FEIFAN ZHOU ◽  
DA XING ◽  
WEI R. CHEN
Keyword(s):  
Cell Death ◽  
Mitochondrial Membrane ◽  
Laser Scanning ◽  
Cell Imaging ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Cellular Event ◽  
Single Cell Imaging ◽  
Induced Apoptosis

Apoptosis is an important cellular event that plays a key role in the therapy of many diseases. The mechanism of the initiation and regulation of photodynamic therapy (PDT)–induced apoptosis is complex. Our previous study found that Photofrin was localized primarily in mitochondria, the primary targets of Photofrin-PDT. The key role of Bax in the mitochondria-mediated apoptosis has been demonstrated in many systems. In order to determine the role of Bax in the mitochondrion-mediated apoptosis induced by Photofrin-PDT, we used the GFP-Bax plasmid to monitor the dynamics of Bax activation after PDT treatment. With laser scanning confocal microscopy, we found that Bax did not translocate from the cytosol to mitochondria when the mitochondrial membrane potential (ΔΨm) disappeared, measured by TMRM. Thus, for Photofrin-PDT, the commitment to cell death is independent of Bax activation.

Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos

Development ◽  
2000 ◽  
Vol 127 (9) ◽  
pp. 1767-1787 ◽  
Author(s):  
V.E. Foe ◽  
C.M. Field ◽  
G.M. Odell
Keyword(s):  
Laser Scanning ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Cycle Phase ◽  
Filamentous Actin ◽  
Mitotic Apparatus ◽  
Scanning Confocal Microscopy ◽  
Drug Treatments

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.

Meiotic spindle organization in fertilized Drosophila oocyte: presence of centrosomal components in the meiotic apparatus

1996 ◽  
Vol 109 (5) ◽  
pp. 911-918 ◽  
Author(s):  
M.G. Riparbelli ◽  
G. Callaini
Keyword(s):  
Confocal Microscopy ◽  
Indirect Immunofluorescence ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Equatorial Region ◽  
Meiotic Spindle ◽  
Scanning Confocal Microscopy ◽  
Bipolar Spindles ◽  
Drosophila Embryos ◽  
Egg Cortex

We examined spindle reorganization during the completion of meiosis in fertilized and unfertilized oocytes of Drosophila using indirect immunofluorescence and laser scanning confocal microscopy. The results defined a complex pathway of spindle assembly during resumption of meiosis, and revealed a transient array of microtubules radiating from the equatorial region of the spindle towards discrete foci in the egg cortex. A monastral array of microtubules was observed between twin metaphase II spindles in fertilized and unfertilized eggs. The microtubules originated from disk-shaped material stained with Rb188 antibody specific for an antigen associated with the centrosome of Drosophila embryos. The Drosophila egg, therefore, contains a maternal pool of centrosomal components undetectable in mature inactivated oocytes. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles.

scholarly journals MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

2018 ◽  
Vol 51 (2) ◽  
pp. 793-811
Author(s):  
Qiang Tong ◽  
Ying Zhu ◽  
Dandan Zhang ◽  
Qing Cai ◽  
Wenchun Qu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Key Factors ◽  
Argonaute 2 ◽  
Scanning Confocal Microscopy ◽  
Imaging Flow Cytometry ◽  
Microscopy Method ◽  
Stimulation Of

Background/Aims: MicroRNA (miRNA)-induced suppression of dendritic cells (DCs) has been implicated in many diseases. Therefore, accurate monitoring of miRNA endocytosis by DCs is important for understanding the role of miRNAs in many diseases. Recently, a method for measuring the co-localization of Argonaute 2 (AGO2)-associated miRNAs on laser-scanning confocal microscopy method was proposed to localize the miRNAs. But its definition was limited by the number of observed cells through its accuracy. Methods: In this study, a method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs. miR-590-5p proven to play an important role in tumor immunity. This method enabled the quantification, visualization and localization of the fluorescence intensity in 30,000 individual cells. Results: Using this method, the DCs with different endocytotic ability were distinguished. The behaviour of miR-590-5p during endocytosis under the stimulation of tumor antigen in DCs was observed, binding to its cognate target mRNA and degradation in DCs. Conclusion: This method based on imaging flow cytometry provide an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.

Integrin α2β1 (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1804-1809 ◽  
Author(s):  
Joachim Werr ◽  
Joakim Johansson ◽  
Einar E. Eriksson ◽  
Per Hedqvist ◽  
Erkki Ruoslahti ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Critical Role ◽  
Collagen Gel ◽  
Time Lapse ◽  
Surface Expression ◽  
Laser Scanning Confocal Microscopy ◽  
Rat Mesentery

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the 2β1 (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the 2β1 integrin reduced PMN migration velocity in the extravascular tissue by 73% ± 3% and 70% ± 10%, respectively ( means ± SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-2 mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the 4β1 or 5β1integrins. Further evidence for a critical role of expression of 2β1 integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-2 mAb treatment. Thus, expression of 2β1 integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated.

scholarly journals The role of Ca2+-activated Cl− current in tone generation in the rabbit corpus cavernosum

2017 ◽  
Vol 313 (5) ◽  
pp. C475-C486 ◽  
Author(s):  
Karen I. Hannigan ◽  
Caoimhin S. Griffin ◽  
Roddy J. Large ◽  
Gerard P. Sergeant ◽  
Mark A. Hollywood ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Corpus Cavernosum ◽  
Laser Scanning Confocal Microscopy ◽  
Isometric Tension ◽  
Scanning Confocal Microscopy ◽  
Rabbit Corpus Cavernosum ◽  
Intracellular Ca ◽  
Low Sensitivity ◽  
Tone Generation

Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl− ( IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa. T16Ainh-A01 and CaCCinh-A01 (10 μM) reduced IClCa by ~85%, while 30 μM abolished it. L-type Ca2+ current was unaffected by 10 μM CaCCinh-A01 but was reduced by 50% at 30 μM CaCCinh-A01, 46% at 10 μM T16Ainh-A01, and 78% at 30 μM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60–70% at 10 μM and >90% at 30 μM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 μM, CaCCinh-A01; 14 μM, T16Ainh-A01). CaCCinh-A01 at 10 μM had little effect on 60 mM KCl contractures, though they were reduced by 30 μM CaCCinh-A01 and T16Ainh-A01 (10 μM and 30 μM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 μM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.

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