Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods

1979 ◽  
Vol 37 (1) ◽  
pp. 323-347
Author(s):  
A. Forer ◽  
W.T. Jackson
Keyword(s):  
Phase Contrast ◽  
Close Association ◽  
Rapid Expansion ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Contrast Microscopy ◽  
One Step ◽  
Triton X ◽  
Spindle Structure ◽  
Spindle Fibres

We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form ‘decorated’ filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.

scholarly journals Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions.

1986 ◽  
Vol 102 (5) ◽  
pp. 1832-1842 ◽  
Author(s):  
T Volberg ◽  
B Geiger ◽  
J Kartenbeck ◽  
W W Franke
Keyword(s):  
Plasma Membrane ◽  
Phase Contrast ◽  
Culture Medium ◽  
External Surface ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Immunofluorescent Localization ◽  
Contrast Microscopy ◽  
Perinuclear Cytoplasm ◽  
Micromolar Range

EGTA-induced depletion of Ca2+ ions from the culture medium of Madin-Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin-containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase-contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin-containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin-containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures.

scholarly journals The Occurrence of Intercellular Bridges in Groups of Cells Exhibiting Synchronous Differentiation

1959 ◽  
Vol 5 (3) ◽  
pp. 453-460 ◽  
Author(s):  
Don W. Fawcett ◽  
Susumu Ito ◽  
David Slautterback
Keyword(s):  
Phase Contrast ◽  
Fruit Fly ◽  
Giant Cells ◽  
Electron Microscopic ◽  
Free Communication ◽  
Intercellular Bridges ◽  
Daughter Cells ◽  
Contrast Microscopy ◽  
Undifferentiated Cells ◽  
Multinucleate Giant Cells

A previous electron microscopic study of the cat testis revealed that spermatids derived from the same spermatogonium are joined together by intercellular bridges. The present paper records the observation of similar connections between spermatocytes and between spermatids in Hydra, fruit-fly, opossum, pigeon, rat, hamster, guinea pig, rabbit, monkey, and man. In view of these findings, it is considered likely that a syncytial relationship within groups of developing male germ cells is of general occurrence and is probably responsible for their synchronous differentiation. When clusters of spermatids, freshly isolated from the germinal epithelium are observed by phase contrast microscopy, the constrictions between the cellular units of the syncytium disappear and the whole group coalesces into a spherical multinucleate mass. The significance of this observation in relation to the occurrence of abnormal spermatozoa in semen and the prevalence of multinucleate giant cells in pathological testes is discussed. In the ectoderm of Hydra, the clusters of cnidoblasts that arise from proliferation of interstitial cells are also connected by intercellular bridges. The development of nematocysts within these groups of conjoined cells is precisely synchronized. Both in the testis of vertebrates and the ectoderm of Hydra, a syncytium results from incomplete cytokinesis in the proliferation of relatively undifferentiated cells. The intercellular bridges between daughter cells are formed when the cleavage furrow encounters the spindle remnant and is arrested by it. The subsequent dissolution of the spindle filaments establishes free communication between the cells. The discovery of intercellular bridges in the two unrelated tissues discussed here suggests that a similar syncytial relationship may be found elsewhere in nature where groups of cells of common origin differentiate synchronously.

scholarly journals Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.

1983 ◽  
Vol 97 (4) ◽  
pp. 963-973 ◽  
Author(s):  
P C Letourneau
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Electron Microscopic Observation ◽  
Neurite Growth ◽  
Growth Cones ◽  
Chick Embryos ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Triton X

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

scholarly journals Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation

2003 ◽  
Vol 185 (17) ◽  
pp. 5306-5309 ◽  
Author(s):  
Samuel Amiteye ◽  
Kazuo Kobayashi ◽  
Daisuke Imamura ◽  
Shigeo Hosoya ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Phase Contrast ◽  
Dipicolinic Acid ◽  
Diacylglycerol Kinase ◽  
Phase Contrast Microscopy ◽  
Electron Microscopic ◽  
Contrast Microscopy ◽  
Microscopic Studies ◽  
Essential Function ◽  
Kinase Homologue

ABSTRACT The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

Actin in spindles of Haemanthus katherinae endosperm. II. Distribution of actin in chromosomal spindle fibres, determined by analysis of serial sections

1979 ◽  
Vol 37 (1) ◽  
pp. 349-371
Author(s):  
A. Forer ◽  
W.T. Jackson ◽  
A. Engberg
Keyword(s):  
Functional Role ◽  
Spindle Pole ◽  
Serial Sections ◽  
Heavy Meromyosin ◽  
A Cell ◽  
Spindle Microtubules ◽  
Spindle Structure ◽  
Spindle Fibres

We have studied the arrangements of actin-containing filaments in 13 bundles of kinetochore microtubules in glycerinated, heavy meromyosin-treated Haemanthus endosperm cells: 7 bundles were in a cell at anaphase, and 6 were in a cell at metaphase. Actin-containing filaments were present in each of the 13 bundles of kinetochore microtubules: they were in amongst the microtubules in the bundle and seemed to be associated with the microtubules. Actin-containing filaments in each bundle seemed to terminate at the kinetochores. Actin-containing filaments associated with the kinetochore microtubules were of consistent polarity (the arrowheads pointed towards the kinetochores) whereas those associated with other microtubles and those not associated with microtubules did not have consistent polarity (some pointed towards the spindle pole, others pointed away from it). Roughly, there were as many individual stretches of actin-containing filaments identified per bundle of kinetochore microtubules as there were microtubules which terminated at the kinetochore. These data suggest that actin-containing filaments in spindles have a functional role. We used 2 glycerination procedures in our studies (one for each cell), and neither seemed to disrupt the basic microtubule arrangements: the arrangements of spindle microtubules seen after glycerination of Haemanthus endosperm were identical to those described previously by others in non-glycerinated glutaraldehyde-fixed Haemanthus endosperm. Thus we argue that spindle structure is not disrupted by the procedures, and therefore that the arrangements of actin-containing filaments are not artifacts of the glycerination procedures. The only difference between microtubules in glycerinated cells and microtubules in untreated cells is that there seem to be fewer in the glycerinated cells. The possible role of actin-containing filaments in the spindle is discussed.

The origin and fate of the nuclear membrane in meiosis

1960 ◽  
Vol 152 (948) ◽  
pp. 353-366 ◽  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Phase Contrast ◽  
Close Association ◽  
Early Prophase ◽  
Daughter Cells ◽  
Nuclear Membranes ◽  
Contrast Microscopy ◽  
Surrounding Membrane

The behaviour of the nuclear membrane during meiotic division in locust spermatocytes has been studied by electron microscopy. Preliminary observations were made on living cells from locusts, grasshoppers and beetles by phase-contrast microscopy and on snail and newt spermatocytes by electron microscopy. The mitochondria come into close association with the nuclear membrane during prophase and are often clustered round regions of degenerating nuclear membrane. The mitochondrial membranes sometimes appear to be fused with the nuclear membrane, and gaps in the latter are often found near mitochondria. Lamellar stacks are found in the cytoplasm during early prophase and closely resemble the nuclear membrane in the same cells ; they are generally annulate in spermatogonia and primary spermatocytes butsmooth-walled in secondary spermatocytes. There is evidence that the stacks arise by repeated folding of the nuclear membrane and become converted into endoplasmic reticulum. After division, the daughter cell chromosomes are at first devoid of a surrounding membrane. Elements of the endoplasmic reticulum accumulate between the mitochondria and gradually surround the chromosomes. These elements fuse to form a continuous double nuclear membrane. It is suggested that the nuclear membrane, endoplasmic reticulum and cell membrane are composed of the same material, which can be transformed into different structures according to the needs of the cell. The nuclear membrane is converted into endoplasmic reticulum during division and stored in the cytoplasm . The new nuclear membranes in the daughter cells are formed by the fusion of elements of the endoplasmic reticulum.

Observation of biological macromolecules using various electron microscopic methods

1978 ◽  
Vol 36 (2) ◽  
pp. 170-171
Author(s):  
Mitsuo Ohtsuki ◽  
Michael Sogard
Keyword(s):  
Electron Microscope ◽  
Phase Contrast ◽  
Image Interpretation ◽  
Density Lipoprotein ◽  
Biological Macromolecules ◽  
Contrast Effects ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Structural Investigations ◽  
Staining Techniques

Structural investigations of biological macromolecules commonly employ CTEM with negative staining techniques. Difficulties in valid image interpretation arise, however, due to problems such as variability in thickness and degree of penetration of the staining agent, noise from the supporting film, and artifacts from defocus phase contrast effects. In order to determine the effects of these variables on biological structure, as seen by the electron microscope, negative stained macromolecules of high density lipoprotein-3 (HDL3) from human serum were analyzed with both CTEM and STEM, and results were then compared with CTEM micrographs of freeze-etched HDL3. In addition, we altered the structure of this molecule by digesting away its phospholipid component with phospholipase A2 and look for consistent changes in structure.

Immunocytochemical Localization of Amyloid Protein AA in the “Dense Fibrillar Inclusions” of the Reticuloendothelical Cells

1977 ◽  
Vol 35 ◽  
pp. 438-439
Author(s):  
T. Shirahama ◽  
M. Skinner ◽  
A.S. Cohen
Keyword(s):  
Amyloid Fibril ◽  
Close Association ◽  
Gel Filtration ◽  
Fibril Formation ◽  
Amyloid Protein ◽  
Electron Microscopic ◽  
Amyloid Deposits ◽  
Amyloid Fibril Formation ◽  
Electron Microscopic Technique ◽  
Lysosomal System

A1thought the mechanisms of amyloidogenesis have not been entirely clarified, proteolysis of the parent proteins may be one of the important steps in the amyloid fibril formation. Recently, we reported that "dense fibrillar inclusions" (DFI), which had the characteristics of lysosomes and contained organized fibrillar profiles as well, were observed in the reticuloendothelial cells in close association with the foci of new amyloid deposits. We considered the findings as evidence for the involvement of lysosomal system in amyloid fibril formation (l). In the present study, we attempted to determine the identity of the contents of the DFI by the use of antisera against the amyloid protein (AA) and an immuno-electron microscopic technique.Amyloidosis was induced in CBA/J mice by daily injections of casein (l). AA was isolated from amyloid-laden spleens by gel filtration and antibody to it was produced in rabbits (2). For immunocytochemistry, the unlabeled antibody enzyme method (3) was employed.

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