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PubMed2006

Image correlation algorithm for measuring lymphocyte velocity and diameter changes in contracting microlymphatics.

Efforts have recently been made to estimate wall shear stress throughout the contractile cycle of mesenteric rat lymphatics with a high speed video microscopy system. This was prompted by reports in the literature that lymphatic pumping is related to wall shear stress. While one can estimate wall shear stress by tracking lymphocyte velocity, it is prohibitively tedious to manually track particles over a reasonable time frame for a good number of experiments. To overcome this, an image correlation method similar to digital particle imaging velocimetry was developed and tested on contracting lymphatics to measure both vessel diameter and fluid velocity. The program tracked temporal fluctuations in spatially averaged velocity with a standard error of prediction of 0.4 mm/s. From these studies we have measured velocities ranging from -2 to 4 mm/s. Diameter changes were also measured with a standard error of 7 microm. These algorithms and techniques could be beneficial for investigating various changes in contractile behavior as a function of changes in velocity and wall shear stress.

Algorithms↗
PubMed2005

An integrative model of the self-sustained oscillating contractions of cardiac myocytes.

Computational cell models appear as necessary tools for handling the complexity of intracellular cell dynamics, especially calcium dynamics. However, while oscillating intracellular calcium oscillations are well documented and modelled, a simple enough virtual cell taking into account the mechano-chemical coupling between calcium oscillations and cell mechanical properties is still lacking. Considering the spontaneous periodic contraction of isolated cardiac myocytes, we propose here a virtual cardiac cell model in which the cellular contraction is modelled using an hyperelastic description of the cell mechanical behaviour. According to the experimental data, the oscillating cytosolic calcium concentrations trigger the spatio-temporal variation of the anisotropic intracellular stresses. The finite element simulations of the virtual cell deformations are compared to the self-sustained contractions of isolated rat cardiomyocytes recorded by time-lapse video-microscopy.

Animals↗
PubMed1994

Direct effects of acute administration of 3, 5, 3' triiodo-L-thyronine on myocyte function.

Recent studies have suggested that acute administration of 3, 5, 3' triiodo-L-thyronine (T3) may increase ventricular performance; however, the direct cellular effects of acute T3 administration are not understood. The objectives of this study were to determine (1) whether T3 acts directly on the cardiac muscle cell (myocyte) itself, and (2) whether T3 acts independently of the myocyte beta-adrenergic receptor. Accordingly, isolated myocyte function was examined using video-microscopy in normal porcine myocytes (n = 60) in the control state and in the presence of increasing T3 concentrations (10 to 500 pmol/L). T3 caused myocyte shortening extent, shortening velocity, and lengthening velocity to increase in a dose-dependent manner. For example, shortening velocity increased from 49.2 +/- 4.3 microns/s at baseline to 66.5 +/- 6.1 microns/s with 100 pmol/L T3 (p < 0.05). beta-Adrenergic stimulation with 25 nmol/L isoproterenol increased shortening velocity to 97.6 +/- 5.7 microns/s; isoproterenol with T3 increased shortening velocity further to 168.5 +/- 10.9 microns/s. Analysis of variance revealed that this increase with T3 was independent of and additive to the beta-adrenergic receptor system. In summary, T3 caused a dose-dependent increase in myocyte contractile performance, and these effects were independent of and additive to beta-adrenergic receptor stimulation. Thus, acute T3 administration may provide a novel modality to improve left ventricular contractile function independent of the beta-adrenergic receptor system.

Animals↗
PubMed1983

Tetracaine modifies the fragmentation mode of heated human erythrocytes and can induce heated cell fusion.

It is known that human erythrocytes in saline fragment by development of an unstable surface wave on the cell rim when cells are heated through the denaturation temperature of the structural protein, spectrin. Here the influence of tetracaine on the fragmentation process has been recorded and analysed by video microscopy of cells heated in rectangular glass microcapillaries. The number of waves per cell rim decreases with increasing tetracaine concentration until, at 0.5 mM tetracaine, wave growth on the cell rim is suppressed on most cells and the cells internalize membrane at the cell dimple. The rate constant for the change in the number of waves per cell with increasing tetracaine concentration is 9.6 mM-1 at a heating rate of 0.5 K/s. 50% of heated cells internalize membrane at 0.14 mM tetracaine. When cells are heated rapidly in suspension in test tubes the presence of tetracaine reduces the temperature for 50% haemolysis from 66 degrees C for washed control cells to 60.5 degrees C for cells in 2 mMs tetracaine. Cells heat in microcapillaries in tetracaine concentrations of 3 mM and higher begin to swell before the spectrin denaturation temperature is reached. Cell fusion was observed at and above the spectrin denaturation temperature in cells heated in 3 and 4 mM tetracaine. It was also noted that the morphology of erythrocytes maintained in 3.6 mM tetracaine for times up to 30 min at 37 degrees C or 20 degrees C was strongly dependent on temperature and time.

Cell Fusion↗
PubMed1992

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Mechanisms of mediator release from human skin mast cells upon stimulation by the bradykinin analog, [DArg0-Hyp3-DPhe7]bradykinin.

We have used the bradykinin analog, [DArg0-Hyp3-DPhe7]-bradykinin, as a model stimulus with which to examine peptide-induced mediator release from human skin mast cells (SMC) and to compare it with IgE-mediated release from the same cells. The bradykinin analog was an effective histamine secretagogue, inducing a comparable maximal level of release to that observed for anti-IgE. By contrast to anti-IgE, however, [DArg0-Hyp3-DPhe7]-bradykinin did not stimulate marked release of prostaglandin D2 (PGD2) from these cells. In experiments where cells were exposed to both stimuli simultaneously, histamine release was additive, while PGD2 release was the same as that observed for anti-IgE alone. The kinetics of [DArg0-Hyp3-DPhe7]-bradykinin-stimulated histamine release were rapid, with 50% of maximal release being achieved within 30 sec, compared to 2-3 min for anti-IgE. Interestingly, when both stimuli were applied simultaneously, the kinetics of release were intermediate between those of either stimulus alone. Studies of the signal transduction pathways that may be involved in [DArg0-Hyp3-DPhe7]-bradykinin-induced histamine release revealed striking differences to results obtained with anti-IgE. While agents that increase intracellular cyclic AMP have a pronounced inhibitory effect on IgE-mediated release, forskolin, isobutylmethylxanthine and isoproterenol were all totally ineffective at inhibiting histamine release induced by the bradykinin analog. Similarly, staurosporine, a relatively selective inhibitor of protein kinase C, and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) an activator of this enzyme, both have pronounced effects on IgE-mediated histamine release from SMC but were completely inactive with regard to [DArg0-Hyp3-DPhe7]-bradykinin-stimulated release. SMC stimulated with this peptide showed characteristic changes in intracellular free calcium levels, as assessed by digital video microscopy. This response differs from that induced by anti-IgE in that it had a more rapd onset, achieved a lower peak, and decayed much more rapidly. Analysis at the single cell level showed that cells that responded in this fashion upon exposure to the bradykinin analog were capable of showing an additional response upon subsequent exposure to anti-IgE. We conclude that histamine release from SMC in response to [DArg0-Hyp3-DPhe7]-bradykinin occurs via a completely different mechanism from that in response to IgE-mediated stimuli. Peptide-induced release is rapid and is not susceptible to pharmacologic manipulation of intracellular cyclic AMP or protein kinase C but utilizes a rapid transient shift in intracellular calcium concentrations as part of its signal transduction pathway.

Antibodies, Anti-Idiotypic↗
PubMed1991

Reversal of rapid axonal transport at a lesion: leupeptin inhibits reversed protein transport, but does not inhibit reversed organelle transport.

The hypothesis that the reversal of rapid axonal transport requires a proteolytic conversion of anterograde transport vesicles was examined using sciatic nerve preparations from Xenopus laevis and leupeptin as an inhibitor of proteolysis. The transport of newly synthesized 35S-labeled proteins was studied with a position-sensitive detector of radiation. Organelle transport in isolated myelinated axons was studied by video microscopy. Leupeptin (0.1-0.4 mM) reduced the anterograde-to-retrograde reversal of protein transport adjacent to an axonal lesion. In experiments in which organelle transport was observed close to lesions in axons maintained in a medium compatible with intracellular function, 1.0 mM leupeptin inhibited neither the anterograde-to-retrograde nor the retrograde-to-anterograde reversal of organelle transport. In addition, in experiments in which conditions approximated those used to study protein transport, organelle transport away from the lesion was not inhibited by 1.0 mM leupeptin. A comparison of the morphology of rapidly transported organelles that underwent anterograde transport to the morphology of those that returned from a lesion (with or without the presence of leupeptin) provided no evidence that a morphological conversion was a necessary step in transport reversal.

Animals↗
PubMed1989

A two-step method for permeabilization of Drosophila eggs.

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-van't Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringer's solution.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Propanol↗
PubMed1992

Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte.

The volumetric response of oocytes during rapid alterations of the extracellular osmotic environment were recorded using video microscopy. From these observations, the kinetics of water loss for human and mouse oocytes were determined over the temperature range 37 to 10 degrees C, including 37, 30, 20, and 10 degrees C. The changes in diameter of oocytes were measured over a 5-min period and a computer model was used to derive values for membrane water permeability (Lp) and inactive volume (Vb) and to compare the experimental data to the predicted values. The results for the mouse oocyte Lp were comparable to values determined by other methods. However the human data, for both failed-to-fertilize and fresh oocytes, have a wide range of values with large standard deviations. The Lp values at the various temperatures were used to calculate the Arrhenius activation energy (Ea). An Ea value of 9.48 kcal/mol was found for the fresh mouse oocyte, whereas the activation energy for human oocytes was extremely low, 3.73 kcal/mol for fresh oocytes and 1.93 kcal/mol for failed-to-fertilize oocytes.

Animals↗
PubMed1985

Control of neural crest cell dispersion in the trunk of the avian embryo.

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Differential growth of the branches of a regenerating bifurcate axon is associated with differential axonal transport of organelles.

Axonal trees display differential growth during development or regeneration; that is, some branches stop growing and often retract while other branches continue to grow and form stable synaptic connections. In this study, an in vitro model of differential growth is examined to identify the intracellular events responsible for this phenomenon. When the giant cerebral neuron of Aplysia californica is placed in culture, vigorous growth occurs from the ends of both branches of its bifurcate axon. If an appropriate target neuron is placed next to one branch, growth from that branch is unabated while growth from the other branch is suppressed. The bidirectional fast transport of membranous organelles was examined in the two branches by the use of high-resolution video microscopy. Transport was similar in the branches in the absence of a target cell but was much greater in the growing than in the nongrowing branch when a target was present. Electron microscopic examination of fixed specimens confirmed these findings. Differential growth may be initiated or sustained by a diversion from certain branches of materials used in growth which are supplied by fast axonal transport.

Animals↗
PubMed1988

Increase in intracellular calcium induced by the polycation-coated latex bead, a stimulus that causes postsynaptic-type differentiation in cultured Xenopus muscle cells.

The polycation-coated latex bead is a potent stimulus for the induction of postsynaptic-type differentiation in cultured Xenopus myotomal muscle cells. Specializations characteristic of the neuromuscular junction, such as clusters of acetylcholine receptors and other postsynaptic-specific proteins, develop at the bead-muscle contact. Previous studies have shown that a deprivation of extracellular calcium inhibits the effect of the beads in causing the development of these specializations. This suggests that an increase in intracellular Ca2+ is a necessary condition for the development of this specialization. In this study, we tested whether an increase in intracellular calcium is observable upon the bead-muscle contact. The measurement was carried out on cells loaded with the fluorescent calcium indicator fura-2 AM by digitized video microscopy. When polycation-coated beads were added to cells, an increase in intracellular calcium concentration in the range of 5 to 57% of the resting level was observed within 10 sec after bead-muscle contact. Afterward, the calcium level gradually returned to the resting level with a time course of about 3 min. Uncoated beads, which do not induce the formation of acetylcholine receptor clustering, failed to elicit this calcium transient. Removal of extracellular calcium as well as blocking calcium channels with 50 microM verapamil also suppressed this transient induced by the polycation-coated beads. Both treatments are known to suppress the formation of receptor clusters by these beads. These results suggest that the polycation-coated beads cause an influx of calcium by increasing the membrane conductance to this ion. This process may underlie the signaling of the postsynaptic differentiation.

Animals↗
PubMed1988

Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat.

Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of alpha- and beta-myosin heavy chain (MHC), and M-band localization of M-creatine kinase in the reconstituting heart cells were studied. Myofibril formation was demonstrated by the use of antibodies against either cardiac C-protein or myomesin as early differentiation markers. Four days after plating, small myofibrils could be identified in attached cells in a perinuclear fashion; later in culture the cells displayed various shapes and myofibril distribution. Frequently a patchy distribution of myofibrils within the extending peripheral processes could be observed. Colocalization of sarcomeres and phalloidin-stained F-actin filament bundles was demonstrated by double fluorescence staining and by the use of high intensifying video microscopy and computerized image processing. The immunofluorescence distribution of alpha- and beta-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% alpha-MHC and 70% beta-MHC in rod-shaped cells to about 100% beta-MHC and 70% alpha-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in alpha-MHC at the expense of increasing amounts of beta-MHC with time in culture as assessed by sodium dodecyl sulfate gel electrophoresis of total cell homogenates. In addition, whereas rod-shaped newly isolated cardiomyocytes showed a clear M-band association of M-creatine kinase as found in adult heart tissue, adult cultivated spread out cells did not show a cross-striated pattern after incubation with antibody. Taken together, these observations suggest that adult cardiomyocytes not only undergo extensive morphological transitions in long-term cultures, but also generate new myofibrillar structures lacking M-creatine kinase and containing the beta-MHC, thus fitting the characteristics of fetal myofibrils. These results indicate a change from the adult terminally differentiated to a less differentiated state of the cardiac cells in culture.

Animals↗
PubMed1989

Teloplasm formation in a leech, Helobdella triserialis, is a microtubule-dependent process.

Fertilized eggs of the leech Helobdella triserialis undergo a cytoplasmic reorganization which generates domains of nonyolky cytoplasm, called teloplasm, at the animal and vegetal poles. The segregation of teloplasm to one cell of the eight-cell embryo is responsible for a unique developmental fate of that cell, i.e., to give rise to segmental ectoderm and mesoderm. We have studied the cytoplasmic movements that generate teloplasm using time-lapse video microscopy; the formation and migration of rings of nonyolky cytoplasm were visualized using transmitted light, while the movements of mitochondria into these rings were monitored with epifluorescence after labeling embryos with rhodamine 123, a fluorescent mitochondrial dye. To examine the likelihood that cytoskeletal elements play a role in the mechanism of teloplasm formation in Helobdella, we examined the distribution of microtubules and microfilaments during the first cell cycle by indirect immunofluorescence and rhodamine-phalloidin labeling, respectively. The cortex of the early embryo contained a network of microtubules many of which were oriented parallel to the cell surface. As teloplasm formation ensued, microtubule networks became concentrated in the animal and the vegetal cortex relative to the equatorial cortex. More extensive microtubule arrays were found within the rings of teloplasm. Actin filaments appeared in the form of narrow rings in the cortex, but these varied apparently randomly from embryo to embryo in terms of number, size, and position. The role of microtubules and microfilaments in teloplasm formation was tested using depolymerizing agents. Teloplasm formation was blocked by microtubule inhibitors, but not by microfilament inhibitors. These results differ significantly from those obtained in embryos of the oligochaete Tubifex hattai, suggesting that the presumably homologous cytoplasmic reorganizations seen in these two annelids have different cytoskeletal dependencies.

Actin Cytoskeleton↗
PubMed1994

In vivo effect of naftidrofuryl on 5-hydroxytryptamine-mediated constriction in rat peripheral microcirculation.

Naftidrofuryl is commonly used in treatment of peripheral vascular disease. Its vasodilator action has been partly explained by its inhibitory effect of 5-HT2 receptors on peripheral arteries in vitro. The purpose of this study was to test in vivo whether naftidrofuryl selectively inhibits 5-hydroxytryptamine (5-HT)-mediated constriction of large arterioles in the peripheral microcirculation. This constriction appears to be 5-HT2 receptor-mediated. Three separate protocols were used to test the effects of naftidrofuryl: chronic injection (15 mg/kg, i.p., twice daily for 5-6 days; n = 7), acute intravenous (i.v.) infusion (15 mg/kg over 30 min; n = 7), or topical application (5 x 10(-8) M, n = 6; 5 x 10(-7) M, n = 5; 5 x 10(-6) M, n = 5; 10(-5) M, n = 7). Male Sprague-Dawley rats (145-185 g body weight) were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was prepared for intravital video microscopy. Diameter response of arterioles (70-120 microns) to increasing concentrations of locally applied 5-HT (10(-8)-10(-4) M) was assessed. In rats receiving no drug treatment, 5-HT caused vasoconstriction of arterioles beginning at 10(-6) M and reaching approximately 40% constriction at 10(-4) M. These vasoactive responses were not altered by chronic daily doses or an acute infusion of naftidrofuryl. 5-HT responses obtained with and without naftidrofuryl applied directly into the cremaster-bath also had little effect on the arteriole response at each of the four concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Interactions between endothelin-1 and atrial natriuretic peptide influence cultured chick cardiac myocyte contractility.

We have previously shown that rat atrial natriuretic peptide (ANP) reduces the contractility of cultured, spontaneously beating chick embryo ventricular cells, an effect opposite to that of endothelin-1. Endothelin-1 has been described as a secretagogue for natriuretic peptides in vitro and in vivo. Natriuretic peptides can inhibit endothelin-1 secretion from cultured endothelial cells, suggesting a negative feedback mechanism between endothelial cells and cardiomyocytes. The aim of this study was to determine whether ANP attenuated the endothelin-1-induced increase in myocyte contractility. Using a video-microscopy system we studied the contractility of isolated cultured chick ventricular myocytes in response to endothelin-1, chicken natriuretic peptide (ChNP), and both. We also used Northern blot analysis to study the time course of ChNP expression in response to endothelin-1. Endothelin-1 (10(-8) M) increased chick cardiomyocyte contractility by 20-25% between 5 and 15 min (P < 0.05). Although ChNP (3 x 10(-7) M) did not significantly change the amplitude of contraction in basal conditions, it prevented the endothelin-1-induced increase in contractility (P < 0.05) when perfused prior to endothelin-1, and reversed it when perfused 5 min after endothelin-1 exposure (P < 0.05). Endothelin-1 significantly increased the accumulation of ChNP mRNA in chick ventricular myocytes as early as the 30 min after exposure (P < 0.05), with a maximal effect after 2 h of stimulation (P < 0.01); no effect was observed after 4 h. These data support an interaction between endothelin-1 and natriuretic peptides as autocrine/paracrine factors regulating the contractile function of chick cardiac myocytes, as well as their antagonistic effects on cardiac cell contractility. The early and transient expression of ChNP mRNA in response to endothelin-1 may be involved in this interaction.

Analysis of Variance↗
PubMed1991

Micromanipulation of mitotic chromosomes in PTK2 cells using laser-induced optical forces ("optical tweezers").

To study the potential use of optical forces to manipulate chromosome movement, we have used a Nd:YAG laser at a wavelength of 1.06 microns focused into a phase contrast microscope. Metaphase and anaphase chromosomes were exposed while being monitored by video microscopy. The results indicated that when optical forces were applied to late-moving metaphase chromosomes on the side closest to the nearest spindle pole, the trapped chromosomes initiated movement to the metaphase plate. The chromosome velocities were two to eight times the normal rate depending on the chromosome size, geometry, and trapping site. At the initiation of anaphase, a pair of chromatids could be held by the optical trap and kept motionless throughout anaphase while the other pairs of chromatids separated and moved to opposite spindle poles. As a result, the trapped chromosome either was incorporated into one of the daughter cells or was lost in the cleavage furrow, or the two chromatids eventually separated and moved to their respective daughter cells. If the trap was removed at the beginning of anaphase B, the chromosome moved back to the poles. Our experiments demonstrate that the laser-induced optical force trap is a potential new technique to study noninvasively the mitotic spindle of living cells.

Analysis of Variance↗
PubMed1992

Mammary epithelial reorganization on extracellular matrix is mediated by cell surface galactosyltransferase.

When plated at appropriate densities in serum-free media, the COMMA-D mammary epithelial cell line rapidly reorganizes into multicellular spheres on the basement membrane matrix derived from Engelbreth-Holm-Swarm murine tumor. Using time-lapse video-microscopy, four stages of reorganization were discerned during the first 24 h of culture. In the first few hours, cells attached to the matrix, elongated, migrated, and formed chains. In the next 6 h, chains of cells linked together in anastomosing networks. In the period between 8 and 18 h postplating, the networks contracted, resulting in dense cords radiating from central aggregates. During the final 6 h, the cords were drawn into the aggregates, which condensed further into spheres. The events occurring during mammary epithelial cell reorganization on the matrix were shown to be mediated by cell surface beta-1,4-galactosyltransferase (GalTase), a receptor that binds N-acetylglucosamine residues on glycosylated proteins. GalTase activity was evident at the surface of cells cultured on reconstituted matrix for 3 h but was absent from cells on glass. The protein alpha-lactalbumin (alpha-LA) inhibits the association of GalTase with N-acetylglucosamine. alpha-LA present from the beginning of culture on reconstituted matrix had no effect on cell attachment but caused concentration-dependent inhibition of the first two steps of reorganization, i.e., cell elongation and network formation, which then interfered with subsequent events. These observations were replicated using polyclonal antibodies to GalTase. Reorganization was impaired when alpha-LA was added during the first two stages but no effect was observed when it was added during the last two stages. Cells cultured on plastic, which lack surface GalTase activity, were unperturbed by incubation with alpha-LA. Thus certain events (cell elongation and network elaboration) during mammary epithelial cell reorganization on reconstituted matrix are GalTase dependent, while others (attachment, network contraction, and compaction) are not. The functional and temporal specificity of GalTase involvement indicates that GalTase mediates cell-matrix, but not cell-cell, interactions during epithelial morphogenetic events in culture.

Animals↗