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PubMed1986

Rapid vesicle formation and aggregation in abnormal human biles. A time-lapse video-enhanced contrast microscopy study.

Rapid nucleation of cholesterol crystals has previously been shown to provide a sharp discrimination between abnormal (cholesterol gallstone-associated) and normal human gallbladder bile. In the present study, we sought to further clarify the crystal nucleation process by time-lapse microscopy using a novel high-resolution video-enhanced microscopy technique. Using a previously described method for removal of particles from abnormal biles, we found a strikingly rapid rate of de novo formation of unilamellar vesicles, soon followed by massive vesicular aggregation, culminating in crystal formation. In normal biles, by contrast, this rapid aggregation process was not observed and the isolated unilamellar vesicles showed prolonged stability. Morphometric analysis of interval particle counts showed statistically significant differences. The process of cholesterol monohydrate crystal nucleation in supersaturated human bile is characterized by a sequential combination of vesicle formation, vesicle aggregation, and subsequent crystal formation. The primary distinction between abnormal and normal biles resides only in the consistent rapidity of onset and completion of these events in the abnormal biles.

Bile↗
PubMed2004

Confocal and video imaging of cytoskeleton dynamics in the leech zygote.

Ooplasmic segregation in the late interphase zygote of the leech Theromyzon trizonare is accomplished by reorganization of an ectoplasmic cytoskeleton formed by polar rings and meridional bands. The dynamic properties of this cytoskeleton were explored by time-lapse confocal and video microscopy. Cytoskeleton assembly was investigated in zygotes pulse-labeled with microinjected fluorophore-tagged or biotin-tagged dimeric tubulin and G-actin. Cytoskeleton disassembly was studied by comparing the linear dimensions of the cytoskeleton at different time points during late interphase. The relative distributions of F- and-G-actin were determined after microinjection of rhodamine-labeled actin and fluorescein-labeled DNase I. Results showed that labeled precursors were readily incorporated into a network of microtubules or actin filaments. Bipolar translocation of the rings and meridional bands was accompanied by the rapid assembly and disassembly of microtubules and actin filaments. Because labeled microtubules and microfilaments gradually decreased, the rate of cytoskeleton disassembly was greater than the rate of cytoskeleton assembly. Hence, ooplasmic segregation was accompanied by the rapid turnover of cytoskeletal components. Co-distribution of F- and-G-actin during mid and late interphase may favor polymer-monomer interchange. We conclude that cytoskeleton reorganization during foundation of cytoplasmic domains can be conveniently studied in the live leech zygote after microinjection of labeled precursors.

Actins↗
PubMed1996

Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions.

To explore primate fertilization, oocytes and zygotes from fertile rhesus monkeys were imaged throughout fertilization, polyspermy, and artificial activation using confocal microscopy for microtubules and DNA, as well as ratiometric computer-enhanced video microscopy for intracellular calcium. Unfertilized oocytes displayed microtubules only in the radially oriented meiotic spindles. At insemination, a large calcium transient was followed by a series of smaller oscillations, and sperm astral microtubules had assembled from the sperm centrosome by 2.5 h after transient onset. This aster enlarged, and later duplicated, as the pronuclei converged near the cortex. Pronuclear apposition was prevented by microtubule inhibitors. At mitotic prophase, microtubules ensheathed both sets of condensing chromosomes. At metaphase, the spindle was barrel-shaped and eccentrically positioned with two small asters at the pole with the sperm tail. Microtubules emanating from the telophase spindle interacted with the adjacent cortex and displaced the spindle toward the cell center as first cytokinesis ensued. During polyspermy, each sperm nucleated an aster, and the frequency of calcium oscillations increased. Activation resulted initially in disarrayed microtubules that eventually organized into functional mitotic spindles. These kinetic results demonstrate that rhesus monkeys accomplish fertilization in a fashion nearly identical to that of humans and are, therefore, ideal models in which to investigate cytoskeletal events during human reproduction.

Adenine↗
PubMed1991

Finger microcirculation in classical migraine. A video-microscopic study of nailfold capillaries.

We assessed microcirculation in nailfold capillaries of 22 patients with classical migraine and 22 normal subjects matched for age and sex using video-microscopy. Blood flow measurements were performed under standardized room temperature, after a 3-min warm water bath and after local cooling. Under standardized conditions, skin temperature at the fingertip was significantly lower in patients with migraine. Morphological differences, however, could not be found between patients and controls. After cooling of the nailfold area, blood flow stop due to vasospasm occurred in 18 of the 22 migraine patients in contrast to 1 of the 22 control subjects only. Capillary blood cell velocity was significantly decreased in the migraine group under all test conditions. Drug treatment did not account for the different prevalences of vasospasm in migraine patients and normal controls. Our findings demonstrate that patients with migraine often have subclinical microcirculatory disorders of the upper limbs.

Adolescent↗
PubMed1996

Flow velocity of single lymphatic capillaries in human skin.

The purpose of this study was to investigate the previously unknown flow velocity in single lymphatic capillaries of humans in the supine position. Fifteen healthy subjects (10 women and 5 men; mean age 35.8 +/- 13.1 yr) were studied. Ten microliters of fluorescein isothiocyanate-dextran (150,000 mol wt) were injected into the subepidermal layer of the foot dorsum. The filling of the microlymphatics from the resulting depot was visualized by fluorescence video microscopy and stored on videotape. Flow velocity in the microlymphatics was determined on the video screen by direct measurement of the advancement of dyed lymph during a given time. The following median velocities were obtained: 0.51 mm/s (0.27 and 0.61 mm/s for lower and upper quartiles, respectively) for velocity during initial network filling and 9.7 microns/s (6.9 and 14.2 microns/s for lower and upper quartiles, respectively) for resting velocity at the end of the filling period. Mean lymphatic capillary diameter was 54.8 +/- 8.2 microns, and mean network extension was 8.3 +/- 3.2 mm. The high filling velocities are probably due to increased interstitial pressure and volume caused by dye microinjection, whereas the values measured during the end of network filling seem to approach resting flow velocities.

Adult↗
PubMed1993

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm.

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 microns. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 microns, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1993

Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking.

Lipid analogues and glycosylphosphatidylinositol (GPI)-anchored proteins incorporated in glass-supported phospholipid bilayers (SBL) were coupled to small (30 nm diameter) fluorescent beads whose motion in the liquid phase was tracked by intensified fluorescence video microscopy. Streptavidin (St), covalently attached to the carboxyl modified surface of the polystyrene bead, bound either the biotinylated membrane component, or a biotinylated monoclonal antibody (mAb) directed against a specific membrane constituent. The positions of the beads tethered to randomly diffusing membrane molecules were recorded at 0.2 sec intervals for about 1 min. The mean square displacement (rho) of the beads was found to be a linear function of diffusion time t, and the diffusion coefficient, D, was derived from the relation, rho(t) = 4Dt. The values of D for biotinylated phosphatidylethanolamine (Bi-PE) dispersed in an egg lecithin:cholesterol (80:20%) bilayer obtained by this methodology range from 0.05 to 0.6 micron 2/sec with an average of mean value of D = 0.26 micron 2/sec, similar to the value of mean value of D = 0.24 micron 2/sec for fluorescein-conjugated phosphatidylethanolamine (Fl-PE) linked to St-coupled beads by the anti-fluorescein mAb 4-4-20 or its Fab fragment. These values of D are comparable to those reported for Fl-PE linked to 30 nm gold particles but are several times lower than that of Fl-PE in the same planar bilayer as measured by fluorescence photobleaching recovery, D = 1.3 microns 2/sec. The mobilities of two GPI-anchored proteins in similar SBL were also determined by use of the appropriate biotinylated mAb and were found to be mean value of D = 0.25 and 0.56 micron 2/sec for the decay accelerating factor (DAF, CD55) and the human Fc gamma RIIIB (CD16) receptors, respectively. The methodology described here is suitable for tracking any accessible membrane component.

Animals↗
PubMed1990

Morphological characterization of surface-induced platelet activation.

Morphological changes of platelets activated on glass and dimethyldichlorosilane-treated glass were investigated using video microscopy. The platelet morphological changes were quantified by measuring the area and circularity of spreading platelets. In addition, re-organization of cytoskeletal structures of spread platelets was examined. The effects of precoated albumin and fibrinogen on the platelet spreading kinetics were examined as a function of surface protein concentrations. Results showed that platelet shape changes were very sensitive to the surface concentration of precoated proteins. In general, platelets on fibrinogen-precoated surfaces spread fully to a circular shape and developed an extensive inner filamentous zone. In the presence of albumin on the surface, however, platelets could not spread fully and the development of the inner filamentous zone was very poor. For both albumin and fibrinogen, the maximum effects of precoated proteins on platelet shape changes were observed when the surface protein concentration reached the monolayer concentration.

Adsorption↗
PubMed1998

Platelet adhesion to aortic endothelial cells in vitro after thrombin treatment: observation with video-enhanced contrast microscopy.

Secondary thrombus formation following arterial occlusion is suggested to play an important role in the exacerbation of ischemic organ damage. We investigated the effect of thrombin on endothelial cells from the aspect of morphological changes and induction of platelet adhesion to the endothelial cells. Using a video-enhanced contrast microscopy, we observed human aortic endothelial cells (HAEC) following perfusion of human alpha-thrombin of 1.0 U/ml (n = 7) or vehicle (n = 7) for 30 minutes. The endothelial cells began to shrink 15 minutes after thrombin administration. Gaps between the cells were formed. The cells became rearranged orderly in the same direction 30 minutes later. In another study, following pretreatment with human alpha-thrombin 1.0 U/ml (n = 10) or vehicle (n = 7) for 20 minutes and washout, platelets were perfused over HAEC for 30 minutes. Platelets adhered directly to thrombin-treated endothelial cells and became flat on the endothelial cells. Then other platelets were observed to approach to the flattened platelets and aggregated onto it. After washout of floating platelets, adhesion of platelets was further confirmed. These results suggest that thrombin may be involved in the endothelial damage and formation of platelet thrombi on the endothelial cells after blood flow disturbance.

Aorta↗
PubMed1989

Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing.

We have monitored the mixing of both aqueous intracellular and membrane-bound fluorescent dyes during the fusion of human red blood cells to influenza hemagglutinin-expressing fibroblasts using fluorescence spectroscopy and low light, image-enhanced video microscopy. The water-soluble fluorescent dye, N-(7-nitrobenzofurazan-4-yl)taurine, was incorporated into intact human red blood cells. The fluorescence of the dye in the intact red blood cell was partially quenched by hemoglobin. The lipid fluorophore, octadecylrhodamine, was incorporated into the membrane of the same red blood cell at self-quenching concentrations (Morris, S. J., D. P. Sarkar, J. M. White, and R. Blumenthal. 1989. J. Biol. Chem. 264: 3972-3978). Fusion, which allowed movement of the water-soluble dye from the cytoplasm of the red blood cell into the hemagglutinin-expressing fibroblasts, and movement of octadecylrhodamine from membranes of red blood cell to the plasma membrane of the fibroblasts, was observed by fluorescence microscopy as a spatial relocation of dyes, and monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, fluorescence increased after a delay of about 30 s at 37 degrees C, reaching a maximum within 3 min. The kinetics, pH profile, and temperature dependence were similar for both fluorescent events measured simultaneously, indicating that influenza hemagglutinin-induced fusion rapidly establishes bilayer continuity and exchange of cytoplasmic contents.

Animals↗
PubMed1991

Wound repair of human surface respiratory epithelium.

Surface airway epithelium is frequently injured by noxious inhaled agents, epithelial wound repair may be an important process by which the epithelial barrier integrity is maintained. To evaluate the role of surface airway cells in the wound repair process, we developed an in vitro wounding model of human nasal epithelial respiratory cells in primary culture. Circular wounds were made in the epithelial cell culture by detaching, with a glass capillary, approximately 50 cells from the collagen matrix. Video microscopy and electron microscopy observations demonstrated the contribution of two main events during the repair process: the spreading of the cells at the edge of the wounded surface, and the migration of epithelial cell sheets. Complete wound closure occurred within 5 to 8 h. The inhibition of wound repair by cytoskeleton inhibitors or cellular protein synthesis inhibitors suggested that these factors are involved in the wound repair process of surface airway epithelium.

Actin Cytoskeleton↗
PubMed1990

In vitro pharmacologic separation of corneal endothelial migration and spreading responses.

Repair of corneal endothelial wounds involves two forms of cell translocation: (1) "migration," in which individual cells at the wound edge break contacts with neighboring cells and move as individuals into the wound defect, and (2) "spreading," in which cells within the confluent monolayer adjacent to the wound move as a group into the wound area. The authors combined morphometric analysis of Giemsa-stained cultures, phase-contrast video microscopy, and Rh-phalloidin staining of actin filaments to study the effects of epidermal growth factor (EGF) and indomethacin on the migratory and spreading responses to wounding using an in vitro wound-closure model which mimics the amitotic state and general behavior of human corneal endothelium. They found that EGF stimulated the migration of individual cells from the wound edge, induced cellular elongation, and promoted a diffuse distribution of actin filaments. Indomethacin promoted spreading of the confluent monolayer into the wound defect, induced enlargement and flattening of cells, and promoted the formation of long, thick actin stress fibers. These results provide evidence that the migration and spreading responses of corneal endothelial cells to wounding can be pharmacologically separated. The findings suggest that migration of individual cells during wound repair may result from an endogenous form of EGF-like stimulation and that the elongated shape associated with this form of translocation results, at least in part, from an EGF-like alteration in actin-filament organization. Spreading of the confluent monolayer to cover the wound defect may result from a decrease in cyclic adenosine monophosphate induced by a transient reduction in prostaglandin E2 synthesis. This form of translocation may result, in part, from enlargement and flattening of corneal endothelial cells secondary to an enhancement of actin stress-fiber formation.

Actins↗
PubMed1989

Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons.

The fluorescent lipophilic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was used to examine the distribution of membrane-bound organelles in growth cones of cultured rat sympathetic neurons. Within chemically fixed growth cones, intense DiOC6(3) fluorescence was localized predominately to the base or central region of growth cones. However, in most growth cones several thin DiOC6(3)-fluorescent processes radiated from the base into the periphery, and double fluorescence imaging of single growth cones indicated that these processes were highly colocalized (approximately 79%) with microtubules. The distribution of DiOC6(3) fluorescence in living growth cones was examined using low light-level fluorescence video microscopy. We observed thin fluorescent processes within the periphery of growth cones to undergo length excursions (extension/retraction) and to change orientation (move laterally). During growth cone advance, processes became progressively thicker and were gradually engulfed by the advancing fluorescent mass. When growth cones were viewed with video-enhanced differential interference contrast microscopy, the position of the fluorescent processes correlated with thickened extensions of central-type cytoplasm through which vesiclelike organelle transport often occurred. These observations indicate several features concerning the organization and movement of membranous organelles (MOs) in growth cones: (1) MOs are highly compartmentalized, the majority being localized to the growth cone base; (2) MOs advance into the periphery along distinct pathways probably associated with microtubules; (3) one or more thin continuous MOs, which most likely represent a thin tubular component of the endoplasmic reticulum, generally precedes advance of vesiclelike MOs along individual transport pathways; and (4) transport pathways with their associated MOs are spatially and temporally dynamic.

Animals↗
PubMed2006

Dynamic behaviour of half-sarcomeres during and after stretch in activated rabbit psoas myofibrils: sarcomere asymmetry but no 'sarcomere popping'.

We examined length changes of individual half-sarcomeres during and after stretch in actively contracting, single rabbit psoas myofibrils containing 10-30 sarcomeres. The myofibrils were fluorescently immunostained so that both Z-lines and M-bands of sarcomeres could be monitored by video microscopy simultaneously with the force measurement. Half-sarcomere lengths were determined by processing of video images and tracking the fluorescent Z-line and M-band signals. Upon Ca2+ activation, during the rise in force, active half-sarcomeres predominantly shorten but to different extents so that an active myofibril consists of half-sarcomeres of different lengths and thus asymmetric sarcomeres, i.e. shifted A-bands, indicating different amounts of filament overlap in the two halves. When force reached a plateau, the myofibril was stretched by 15-20% resting length (L0) at a velocity of approximately 0.2 L0 s(-1). The myofibril force response to a ramp stretch is similar to that reported from muscle fibres. Despite the approximately 2.5-fold increase in force due to the stretch, the variability in half-sarcomere length remained almost constant during the stretch and A-band shifts did not progress further, independent of whether half-sarcomeres shortened or lengthened during the initial Ca2+ activation. Moreover, albeit half-sarcomeres lengthened to different extents during a stretch, rapid elongation of individual sarcomeres beyond filament overlap ('popping') was not observed. Thus, in contrast to predictions of the 'popping sarcomere' hypothesis, a stretch rather stabilizes the uniformity of half-sarcomere lengths and sarcomere symmetry. In general, the half-sarcomere length changes (dynamics) before and after stretch were slow and the dynamics after stretch were not readily predictable on the basis of the steady-state force-sarcomere length relation.

Animals↗
PubMed2006

Structural and developmental studies of Chlamydomyzium oviparasiticum from Rhabditis nematodes and in culture.

The oomycete (Peronosporomycete) Chlamydomyzium oviparasiticum, previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the 'achlyoid' pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.

Animals↗
PubMed1997

Locomotory phenotypes of human tumor cell lines and T lymphocytes in a three-dimensional collagen lattice.

Active cellular locomotion is a feature of such diverse cell types as lymphocytes and cancer cells. The locomotory phenotype of a cell should ultimately reflect the biochemical basis of different migratory strategies. We investigated the locomotory behavior of five epithelial cell lines and one non-epithelial human cell-line as well as human CD4+ T lymphocytes in a three-dimensional collagen type I matrix using time-lapse video microscopy and computer assisted cell-tracking. Migration velocity was up to 70 times lower in tumor cells (0.1-0.3 microm/min) as compared to T lymphocytes (7-7.5 microm/min), whereas the percentage of spontaneously active cells was up to twice as high in tumor cells (80-90%) in comparison to T lymphocytes (54%). Persistence, i.e. the degree of roaming, varied appreciably between the different cell types. In conclusion, velocity and persistence may describe distinct migration strategies in different cell types, i.e. discerning T cell migration from tumor cell invasion.

Cell Movement↗
PubMed2005

Propofol-induced calcium signalling and actin reorganization within breast carcinoma cells.

BACKGROUND AND OBJECTIVE: MDA-MB-468 breast carcinoma cells respond to non-volatile anaesthetics such as propofol with an increased migration. Here we investigated the relationship between GABA-A receptor modulators, the mode of calcium oscillation and actin reorganization with regard to breast carcinoma cell migration. METHODS: Expression of the GABA-A receptor was determined by Western blot analysis. Calcium-imaging experiments of individual MDA-MB-468 cells as well as visualization of the F-actin distribution were performed by confocal laser scanning microscopy. Cell migration was investigated in a three-dimensional collagen matrix by time-lapse video microscopy. The GABA agonist propofol was used in a final concentration of 6 microg mL(-1). GABA-A receptor antagonist bicuculline (50 micromol) and selective L-type calcium channel blocker verapamil (5 micromol) were used to modulate the propofol effects. RESULTS: A functional GABA-A receptor is expressed by MDA-MB-468 cells. Activation with propofol resulted in sustained increased intracellular calcium concentrations concomitant with actin reorganization and induction of migration in MDA-MB-468 cells. These propofol effects were completely blocked by verapamil. Spontaneous migration of MDA-MB-468 cells (64.4 +/- 7.0%) was significantly increased by propofol to 85.0 +/- 5.0%. MDA-MB-468 cells co-treated with propofol and verapamil showed a migratory activity of 63.0 +/- 2.0% indicating that verapamil blocked the propofol effect. Similar results were achieved with the GABA-A receptor inhibitor bicuculline (control: 56.3 +/- 8.5%; propofol: 80.5 +/- 7.1%; propofol + bicuculline: 52.5 +/- 8.6%). CONCLUSION: Activation of GABA-A receptor by propofol correlated with an increased migration of MDA-MB-468 breast carcinoma cells, mediated by calcium influx via L-type calcium channels and reorganization of the actin cytoskeleton.

Actins↗
PubMed1989

Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

Guanosine Diphosphate↗