scholarly journals Effects of elevated intracellular magnesium on cytoskeletal integrity

1988 ◽  
Vol 89 (3) ◽  
pp. 321-329
Author(s):  
A.R. Prescott ◽  
J.G. Comerford ◽  
R. Magrath ◽  
N.J. Lamb ◽  
R.M. Warn
Keyword(s):  
Immunofluorescence Microscopy ◽  
Living Cells ◽  
Magnesium Concentration ◽  
Free Calcium ◽  
Actin Microfilaments ◽  
Microtubule Network ◽  
Intracellular Magnesium ◽  
Sham Injection ◽  
Image Intensification ◽  
Tubulin Antibodies

Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These effects were studied using immunofluorescence microscopy of fixed cells, and also in living cells using rhodamine-tubulin or rhodamine-conjugated anti-tubulin antibodies and image intensification and enhancement techniques. Simultaneously and accompanying the disassembly of the microtubule arrays the F-actin stress fibres also disappeared, usually leaving the peripheral and perinuclear F-actin microfilaments intact. In contrast, increasing intracellular magnesium appeared to have no effect on the vimentin-containing intermediate filaments of PtK2 cells. These effects on the cytoskeleton were specific to magnesium and could not be mimicked by either microinjection of injection buffer of equivalent ionic strength or sham injection. Raising the intracellular free calcium to the same extent resulted in the disassembly of the microtubule network, but appeared to have no effect on the F-actin stress fibres.

Variations in the distribution and migration of centriole duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy

1980 ◽  
Vol 43 (1) ◽  
pp. 177-194 ◽  
Author(s):  
J.E. Aubin ◽  
M. Osborn ◽  
K. Weber
Keyword(s):  
Nuclear Membrane ◽  
Immunofluorescence Microscopy ◽  
Cytoplasmic Microtubule ◽  
Migration Patterns ◽  
Large Variability ◽  
Membrane Breakdown ◽  
Large Populations ◽  
Tubulin Antibodies ◽  
Cytoplasmic Microtubules ◽  
And Migration

The localization and migration of centriole duplexes have been studied in PtK2 cells by indirect immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated the usefulness of the immunofluorescence technique to quantitate studies of centriole migration and concomitant events such as cytoplasmic microtubule breakdown in large populations of cells. Centriole duplex locations in normal and Colcemid-treated interphase populations have been compared with duplex locations in prophase cells. A higher percentage of duplexes were found close to the nucleus in prophase than in interphase cells, but approximately 5% of the duplexes remained in the cytoplasm far removed from the nucleus in prophase and throughout the course of duplex separation. Duplex separation occurred along a wide variety of paths and duplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur. Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindles forming far to the side of the condensing chromosomes. The timing of duplex separation did not always coincide either with chromosome condensation or with nuclear membrane breakdown, and in a small percentage of the cells separation occurred as late as prometaphase. These data suggest that normal spindle formation can occur despite the large variability in initial and final centriole duplex location, their migration patterns, and the timing of the different events. Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase; the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.

scholarly journals Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

1978 ◽  
Vol 77 (3) ◽  
pp. R27 ◽  
Author(s):  
M Osborn ◽  
RE Webster ◽  
K Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Immunofluorescence Microscopy ◽  
Uranyl Acetate ◽  
Tubulin Antibodies ◽  
Ptk2 Cell ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Microtubular Structures

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

scholarly journals Spherocytic Hemolytic Disease During Magnesium Deprivation in the Rat

Blood ◽  
1971 ◽  
Vol 38 (4) ◽  
pp. 468-478 ◽  
Author(s):  
MARTIN M. OKEN ◽  
MARSHALL A. LICHTMAN ◽  
DENIS R. MILLER ◽  
PIERRE LEBLOND
Keyword(s):  
Magnesium Deficiency ◽  
Lactate Production ◽  
Male Rats ◽  
Magnesium Concentration ◽  
Red Cell ◽  
Cell Deformability ◽  
Red Cell Deformability ◽  
Intracellular Magnesium ◽  
Erythroid Hyperplasia

Abstract Young male rats (115 g) were maintained on diets containing 4-8 mg of magnesium per 100 g of diet for 12 wk. By 3 wk the characteristic features of magnesium deprivation developed, including decreased plasma and tissue magnesium concentration, growth retardation, ruffled fur, patchy dermatitis, irritability, hyperemia of acral parts, onychymegaly, and in the most severely restricted, premature death. By 7 wk of deprivation, evidence of a hemolytic state existed and thereafter reticulocytosis, spherocytosis, shortened 51Cr red cell survival, erythroid hyperplasia of the bone marrow, and mild anemia were present. Erythrocytes during magnesium deficiency were characterized by decreased intracellular magnesium, glucose utilization, lactate production, ATP and 2,3-DPG concentration. A progressive decrease in red cell deformability as measured by cell elastimetry occurred. The reduction in lactate production and in ATP concentration due to magnesium deficiency may be causal in the development of rigid spherocytes with shortened survival in vivo. In addition, the shape and deformability alteration of the red cell may be due to defective membrane construction in a magnesium-deficient environment.

scholarly journals Microinjection of fluorescent tubulin into dividing sea urchin cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1249-1254 ◽  
Author(s):  
P Wadsworth ◽  
R D Sloboda
Keyword(s):  
Sea Urchin ◽  
Daughter Cell ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Lytechinus Variegatus ◽  
Linear Polymers ◽  
Spindle Poles ◽  
Image Intensification ◽  
Fluorescent Polymer

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.

Determination of Intracellular Magnesium by an Automatic Titration Procedure

1960 ◽  
Vol 6 (6) ◽  
pp. 564-571 ◽  
Author(s):  
Donald A Sones ◽  
Warren F McGuckin ◽  
Khalil G Wakim
Keyword(s):  
Magnesium Concentration ◽  
Normal Range ◽  
Intracellular Magnesium ◽  
Titration Procedure ◽  
Term Administration ◽  
Original Procedure ◽  
Increase In Accuracy ◽  
Automatic Titration

Abstract Our experiences with the automatic titration method of quantitating serum calcium and magnesium are presented. In addition, modifications of the original procedure to allow determination of intraerythrocyte concentrations of magnesium are described. It appears that adoption of a separate magnesium standard in addition to the calcium standard, plus certain other minor modifications designed to minimize the variables inherent in the procedure, provides a significant increase in accuracy and reproducibility of results. Values for intraerythrocyte magnesium concentration in 9 normal dogs were found to range from 4.69 to 5.77 mg./100 ml. Values for 4 dogs that received long-term administration of chlorothiazide also fell within this normal range.

scholarly journals In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.

1984 ◽  
Vol 98 (6) ◽  
pp. 2091-2106 ◽  
Author(s):  
I I Singer ◽  
D W Kawka ◽  
D M Kazazis ◽  
R A Clark
Keyword(s):  
Granulation Tissue ◽  
Wound Repair ◽  
Immunofluorescence Microscopy ◽  
Actin Microfilaments ◽  
Surface Adhesion ◽  
Microfilament Bundles ◽  
Binding Surface ◽  
Double Label ◽  
Fibroblast Adhesion

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.

scholarly journals Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Adrian Müller-Deku ◽  
Joyce C. M. Meiring ◽  
Kristina Loy ◽  
Yvonne Kraus ◽  
Constanze Heise ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Biological Response ◽  
Living Cells ◽  
Optical Control ◽  
Microtubule Cytoskeleton ◽  
Microtubule Network ◽  
Cellular Processes ◽  
Non Invasive ◽  
Chemical Inhibitors ◽  
Neuronal Physiology

Abstract Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

Measurement of [Ca] in single cells and pH in single organelles by fluorescence microscopy

1988 ◽  
Vol 46 ◽  
pp. 46-47
Author(s):  
F.R. Maxfield ◽  
M.L. Shelanski
Keyword(s):  
Single Cells ◽  
Living Cells ◽  
Free Calcium ◽  
Ion Concentrations ◽  
Calcium Indicator ◽  
Controlled Illumination ◽  
Computer Controlled ◽  
Temporal And Spatial ◽  
Intracellular Sorting ◽  
Indicator Dyes

Microscope spectrofluorometry and digital image processing provide the ability to study changes in ion concentrations in living cells with high temporal and spatial resolution. We have used fluorescein labeled macromolecules to measure the pH of specific endosomal compartments (1-3). The ratio of fluorescence intensities at 450 nm and 490 nm excitation provides a measure of the pH (4). The acidification of endosomes detected by this technique provide an explanation for endosome functions including intracellular sorting of ligands and receptors, release of iron from transferrin, and penetration of viruses and toxins into the cytosol (3).Using the tetracarboxylate calcium indicator dyes synthesized by R. Tsien and his colleagues (5), the same instruments can be used for measuring intracellular free calcium, [Ca2+]i, in single cells. We have used the system to measure [Ca2+]i changes associated with cell motility (6-9).Cells are examined using a Leitz Diavert microscope with a computer-controlled illumination and photometry system.

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