Microtubule polarity in taxol-treated isolated spindles

1985 ◽  
Vol 63 (6) ◽  
pp. 519-532 ◽  
Author(s):  
Leah T. Haimo
Keyword(s):  
Electron Microscopy ◽  
Cell Lysis ◽  
Electrophoretic Analysis ◽  
Equatorial Region ◽  
Equatorial Plate ◽  
Spindle Microtubule ◽  
Microtubule Polarity ◽  
Uniform Polarity

Spindles were isolated from Spisula oocytes in the presence or absence of taxol to determine if cell lysis procedures previously used to analyze spindle microtubule polarity may have resulted in the loss of any microtubules of a particular polarity. Electrophoretic analysis indicated that spindle preparations isolated in the presence of taxol contained considerably more tubulin than did those isolated in the absence of taxol. Although there was no corresponding increase in the birefringence of the taxol-treated isolated spindles, thereby suggesting that they did not possess a greater concentration of aligned microtubules between the poles and the equatorial plate, electron microscopy revealed that the taxol-treated isolated spindles did, in fact, contain more microtubules than did the nontaxol-treated isolated spindles. These microtubules were predominantly localized immediately around the centrioles, suggesting that assembly may have occurred during the lysis into taxol. The spindles were decorated with Chlamydomonas dynein to display microtubule polarity. The majority of microtubules, located between the chromosomes and poles, possessed a uniform polarity in both taxol- and nontaxol-treated isolated spindles. In the region of the chomosomes, however, up to 45% of the microtubules were oriented with a polarity opposite to the remainder and were intermingled among them. It is probable that microtubules with opposite polarities arise from the opposing poles and overlap in the equatorial region. The extent of overlap appears to be quite substantial and thus may be of significance during mitotic movements.

scholarly journals Single-cell lysis for visual analysis by electron microscopy

2013 ◽  
Vol 183 (3) ◽  
pp. 467-473 ◽  
Author(s):  
Simon Kemmerling ◽  
Stefan A. Arnold ◽  
Benjamin A. Bircher ◽  
Nora Sauter ◽  
Carlos Escobedo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Cell ◽  
Visual Analysis ◽  
Cell Lysis

Changes Induced by Pre-Fixation in Polycation Bacteriolysis and Surface Alterations Matching the Model of Wall Picnosis in Cell Lysis

1980 ◽  
Vol 35 (9-10) ◽  
pp. 805-810
Author(s):  
Alexandru Popescu ◽  
Stefan Antohi ◽  
Stefan Trasculescu ◽  
Adriana Aurescu ◽  
Nicolae Manolescu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Cell Lysis ◽  
Cell Sensitivity ◽  
Lytic Effect ◽  
Shape And Size ◽  
Scaning Electron Microscopy

Abstract Pretreatments of B. subtilis and S. aureus cells with lower concentrations of fixative agents, led to modifications in bacteriolytic effect exerted by polyarginine and protamine: Glutaraldehyde blocked polycation bacteriolysis while formaldehyde and osmium tetroxide (OSO4) having no influence on polyarginine action, increased constantly the cell sensitivity to protamine in lower doses otherwise nonlytic; the sensitizing action also resulted in the extension of protamine bacteriolytic pattern including several staphylococcal strains; higher bacteriolytic doses of protamine were contrastively unable to lyse OSO4 prefixed cells and gave an inconstant lytic value with formaldehyde treated bacteria. With higher concentrations, OSO4 preserved intactly its sensitizing action while formaldehyde displayed a decrease in its ability to sensitize B. subtilis cells to the lytic effect of protamine. Scaning electron microscopy of polycation treated cells showed prelytic lesions as surface granulations, shape and size modifications and cell splits. The interpretation of the results in terms of intra-and intermolecular adducts accompanied by con­ formational changes in surface macromolecules is discussed. It is concluded that the results match the model of polycation bacteriolysis by wall multizonal picnosis leading to surface splits and thereby triggering cell-lysis.

scholarly journals Daptomycin Exerts Bactericidal Activity without Lysis of Staphylococcus aureus

2008 ◽  
Vol 52 (6) ◽  
pp. 2223-2225 ◽  
Author(s):  
Nicole Cotroneo ◽  
Robert Harris ◽  
Nancy Perlmutter ◽  
Terry Beveridge ◽  
Jared A. Silverman
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Division ◽  
Bactericidal Activity ◽  
Membrane Integrity ◽  
Cell Lysis

ABSTRACT The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

scholarly journals Banding and polarity of actin filaments in interphase and cleaving cells.

1980 ◽  
Vol 86 (2) ◽  
pp. 568-575 ◽  
Author(s):  
J M Sanger ◽  
J W Sanger
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Actin Filaments ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Cleavage Furrow ◽  
Heavy Meromyosin ◽  
Glutaraldehyde Fixation ◽  
Microfilament Bundles ◽  
Uniform Polarity

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.

scholarly journals The Drosophila Kinesin-13, KLP59D, Impacts Pacman- and Flux-based Chromosome Movement

2009 ◽  
Vol 20 (22) ◽  
pp. 4696-4705 ◽  
Author(s):  
Uttama Rath ◽  
Gregory C. Rogers ◽  
Dongyan Tan ◽  
Maria Ana Gomez-Ferreria ◽  
Daniel W. Buster ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Family Member ◽  
Somatic Cells ◽  
Spindle Pole ◽  
The Other ◽  
Chromosome Movement ◽  
Spindle Microtubule ◽  
Important Regulator ◽  
Chromosome Movements

Chromosome movements are linked to the active depolymerization of spindle microtubule (MT) ends. Here we identify the kinesin-13 family member, KLP59D, as a novel and uniquely important regulator of spindle MT dynamics and chromosome motility in Drosophila somatic cells. During prometaphase and metaphase, depletion of KLP59D, which targets to centrosomes and outer kinetochores, suppresses the depolymerization of spindle pole–associated MT minus ends, thereby inhibiting poleward tubulin Flux. Subsequently, during anaphase, loss of KLP59D strongly attenuates chromatid-to-pole motion by suppressing the depolymerization of both minus and plus ends of kinetochore-associated MTs. The mechanism of KLP59D's impact on spindle MT plus and minus ends appears to differ. Our data support a model in which KLP59D directly depolymerizes kinetochore-associated plus ends during anaphase, but influences minus ends indirectly by localizing the pole-associated MT depolymerase KLP10A. Finally, electron microscopy indicates that, unlike the other Drosophila kinesin-13s, KLP59D is largely incapable of oligomerizing into MT-associated rings in vitro, suggesting that such structures are not a requisite feature of kinetochore-based MT disassembly and chromosome movements.

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system

1985 ◽  
Vol 5 (9) ◽  
pp. 2361-2368
Author(s):  
L S Symington ◽  
P Morrison ◽  
R Kolodner
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Electrophoretic Mobility ◽  
Electrophoretic Analysis ◽  
Individual Band ◽  
Agarose Gels ◽  
Cell Extracts ◽  
Recombination System ◽  
Plasmid Recombination

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

scholarly journals Changes in microtubule polarity orientation during the development of hippocampal neurons in culture.

1989 ◽  
Vol 109 (6) ◽  
pp. 3085-3094 ◽  
Author(s):  
P W Baas ◽  
M M Black ◽  
G A Banker
Keyword(s):  
Cell Body ◽  
Hippocampal Neurons ◽  
Regional Differences ◽  
Morphological Characteristics ◽  
The Other ◽  
Developmental Changes ◽  
Microtubule Polarity ◽  
Temporally Correlated ◽  
Uniform Polarity ◽  
Cultured Hippocampal Neurons

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.

Introducing Students to Research: Electron Microscopy of Bacteriophages

2010 ◽  
Vol 18 (4) ◽  
pp. 30-33
Author(s):  
Jorge E. Sanchez ◽  
Erica L. Jacovetty ◽  
Bridget Carragher ◽  
Clinton S. Potter ◽  
Rebecca E. Taurog
Keyword(s):  
Electron Microscopy ◽  
Bacterial Species ◽  
Genetic Material ◽  
Cell Lysis ◽  
Bacterial Host

Bacteriophages, as the name “bacteria-eater” suggests, are viruses that infect bacteria. Bacteriophages, often abbreviated as “phages,” have receptors that bind to specific bacterial species, thus there are many types of bacteriophages. Once a phage interacts with its target bacterium, the phage injects its genetic material into the bacterial host where the phage is replicated to produce many new phages that then leave the host via cell lysis.

scholarly journals Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

1985 ◽  
Vol 5 (9) ◽  
pp. 2361-2368 ◽  
Author(s):  
L S Symington ◽  
P Morrison ◽  
R Kolodner
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Electrophoretic Mobility ◽  
Electrophoretic Analysis ◽  
Individual Band ◽  
Agarose Gels ◽  
Cell Extracts ◽  
Recombination System ◽  
Plasmid Recombination

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.

scholarly journals Chayote Mosaic, a New Disease in Sechium edule Caused by a Tymovirus

Plant Disease ◽  
1997 ◽  
Vol 81 (4) ◽  
pp. 374-378 ◽  
Author(s):  
M. Hord ◽  
W. Villalobos ◽  
A. V. Macaya-Lizano ◽  
C. Rivera
Keyword(s):  
Electron Microscopy ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Particle Morphology ◽  
Electrophoretic Analysis ◽  
New Disease ◽  
Virus Particles ◽  
Sechium Edule ◽  
Leaf Deformation ◽  
Cytological Changes

A sap-transmissible virus was isolated from chayote (Sechium edule) in Costa Rica. Infected plants showed chlorotic spots and rings, and blotchy mosaics, which often coalesced to give a complete mosaic and leaf deformation. By electron microscopy, spherical virus-like particles of approximately 29 nm in diameter were visible, and cytological changes associated with the chloroplasts were observed. The virus particles sedimented in sucrose density gradients as two components, a top component of empty protein shells and a bottom component of electron-dense particles. Electrophoretic analysis showed a single-stranded RNA of approximately 5.7 kb and capsid protein (CP) subunits of ∼22 kDa. The virus was identified as a member of the tymovirus group on the basis of particle morphology, size, sedimentation in sucrose gradients, cytopathological effects, and capsid protein and genome properties, and it was tentatively named chayote mosaic virus (ChMV).

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