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PubMed2001

Activation of Rhoa and ROCK are essential for detachment of migrating leukocytes.

Detachment of the rear of the cell from its substratum is an important aspect of locomotion. The signaling routes involved in this adhesive release are largely unknown. One of the few candidate proteins to play a role is RhoA, because activation of RhoA in many cell types leads to contraction, a mechanism probably involved in detachment. To study the role of RhoA in detachment regulation, we analyzed several subsets of expert migratory leukocytes by video microscopy. In contrast to fast-migrating neutrophils, eosinophils do not detach the rear of the cell unless stimulated with serum. When measuring the amount of active RhoA, with the use of a GST-Rhotekin pulldown assay, we found that serum is an excellent activator of RhoA in granulocytes. Inhibition of RhoA or one of Rho's target proteins, the kinase ROCK, in neutrophils leads to the phenotype seen in eosinophils: the rear of the cell is firmly attached to the substratum, whereas the cell body is highly motile. ROCK-inhibition leads to impaired migration of granulocytes in filters, on glass, and through endothelial monolayers. Also, the ROCK signaling pathway is involved in changes of integrin-mediated adhesion. Eosinophil transduction by a tat-fusion construct containing active RhoA resulted in detachment stimulation in the presence of chemoattractant. From these results we conclude that activation of the RhoA-ROCK pathway is essential for detachment of migratory leukocytes.

Cells, Cultured↗
PubMed1994

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.

Animals↗
PubMed1994

Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape.

Actins↗
PubMed2004

Gene silencing of CENP-E by small interfering RNA in HeLa cells leads to missegregation of chromosomes after a mitotic delay.

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.

Calcium-Binding Proteins↗
PubMed2006

Cortical ventricular zone progenitors and their progeny maintain spatial relationships and radial patterning during preplate development indicating an early protomap.

The graded expression of transcription factors by progenitors in the ventricular zone (VZ) confers positional or area identity that is inherited by subplate (SP) neurons and governs their expression of guidance molecules for thalamocortical axons and other properties required for cortical area specification. This mechanism would be most efficient if VZ progenitors and their SP neuronal progeny maintain neighbor relationships during the generation of the preplate (PP), the precursor of the SP. Therefore, a major goal of this study is to determine whether progenitors in the cortical VZ and their progeny maintain neighbor relationships during the genesis of the neocortical PP. We used time-lapse video microscopy to follow the movements of VZ progenitors and the radial movement of their progeny and distribution in the PP in whole-mount or slice cortical explants from embryonic rats at stages when PP neurons are generated. We show that labeled VZ cells proliferate and have a strong tendency to retain neighbor relationships within the VZ and that their neuronal progeny move superficially along a radial column to form the overlying PP; during this process, their neuronal progeny also retain neighbor relationships and thereby form the PP in spatial register with the VZ progenitors that generate them. This behavior differs from that reported at later stages of cortical development, when cortical plate (CP) neurons are generated, and considerable dispersion is evident among both cells within the VZ and neuronal progeny as they migrate from the VZ to the CP. However, our findings show that at the early stage of cortical development, when PP/SP neurons are generated, the VZ is, at a cellular level, a "protomap" of the PP/SP.

Animals↗
PubMed1993

Mechanisms causing coronary microvascular dysfunction following crystalloid cardioplegia and reperfusion.

OBJECTIVE: The aim was to examine the mechanisms of coronary microvascular dysfunction during cardiopulmonary bypass and ischaemic arrest using a crystalloid cardioplegic solution. METHODS: Porcine hearts were arrested with cold hyperkalaemic (K+ = 25 mmol.litre-1) cardioplegic solution for 1 h during cardiopulmonary bypass and then reperfused for 1 h. Selected hearts were arrested but not reperfused. Coronary vessels of non-instrumented pigs were used as controls. In vitro vascular responses of subepicardial and subendocardial arterioles were examined in a pressurised (40 mm Hg) no flow state with video microscopy. RESULTS: Following 1 h of ischaemic cardioplegia, endothelium dependent relaxations of epicardial arterioles to the receptor mediated agent ADP and the non-receptor-mediated agent calcium ionophore A23187 were moderately reduced, and the contractile responses to KCl or the thromboxane A2 analogue U46619 were reduced compared to responses of vessels from control animals. After 1 h of reperfusion, U46619 caused contraction greater than control values, while contraction to KCl and endothelium dependent relaxations to ADP or A23187 were further reduced. Responses of endocardial microvessels to serotonin were slightly more affected by cardioplegia and reperfusion than were epicardial vessels, while the effect on responses of epicardial and endocardial vessels to bradykinin or A23187 were similar. Endothelium independent relaxation to sodium nitroprusside was not altered in any of the experimental groups. The addition of manganese superoxide dismutase to the cardioplegic solution markedly preserved endothelium dependent responses to ADP and A23187 and contractile response to U46619, compared to the responses of vessels from the plain crystalloid cardioplegia group, but had no effect on relaxation to sodium nitroprusside or on contraction to KCl. Five hours of normokalaemic hypothermia (5-10 degrees C) in Krebs buffer had minimal effect on vasodilator responses. Electron microscopy revealed preserved endothelial and smooth muscle cell structure, and focal mononuclear leucocyte-endothelium adherence following cardioplegic arrest and reperfusion. CONCLUSIONS: Ischaemic cardioplegia-reperfusion induced endothelium dependent and direct smooth muscle microvascular dysfunction is at least partially mediated by prolonged exposure of vessels to hyperkalaemia and to the generation of oxygen derived free radicals. Leucocytes probably mediate injury during reperfusion, while hypothermia has minimal effect on recovery of vasomotor function.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.

Kinesin is a force-generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin-deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.

Biological Transport↗
PubMed2006

Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice.

Type I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons.

Animals↗
PubMed2004

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells.

BACKGROUND: Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). METHODS: Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. RESULTS: Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and alpha-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. CONCLUSIONS: In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Actins↗
PubMed2006

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH.

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.

Abscisic Acid↗
PubMed2006

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI.

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein-DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.

Calcium↗
PubMed2003

Haemodialysis-induced pulmonary granulocyte sequestration in rabbits is organ specific.

BACKGROUND: Haemodialysis (HD) with cuprophan (CU) dialysers leads to a severe transient granulocytopenia. In the present study, we challenge the hypothesis that granulocytes sequester within the pulmonary vasculature simply because this is the first vascular bed encountered. This hypothesis is based upon experiments in which activated plasma or complement fragments were infused into animals, and may not pertain to the more complex HD situation. METHODS: We used a rabbit model of HD, and returned the blood into the caval vein (v-HD) or aorta (a-HD). The mesentery was continuously monitored by intravital video microscopy, whereas other tissues were collected at the nadir of granulocytopenia and analysed immunohistochemically. RESULTS: Compared with controls, the number of granulocytes within alveolar walls was almost 2-fold higher following HD, with no difference between venous and arterial blood return. In addition, both v-HD and a-HD induced granulocyte accumulation within part of the larger pulmonary microvessels, though the amount of granulocytes found was 2-fold higher after v-HD. At no time did a-HD induce granulocyte sequestration within the mesenteric microcirculation. Neither did arterial return increase their number in other first-pass tissues like skeletal muscle or renal glomeruli, but it did so in the liver. In the heart, granulocyte content decreased during HD. CONCLUSIONS: Pulmonary sequestration of granulocytes during CU HD is not simply a first-pass effect, but is organ specific to a great extent. The accumulation within larger microvessels suggests an important role for adhesion molecules, whereas cellular stiffening may be involved in granulocyte retention within alveolar capillaries.

Agranulocytosis↗
PubMed2000

The effect of calcium ionophore A23187 on neurites from embryonic mouse spinal cord explants in culture.

The calcium channel ionophore A23187 has been shown to cause a striking reduction in both the rate of neurite outgrowth and in growth cone motility; it has also been shown that these changes may be reversed when the ionophore is removed from the culture medium. The evidence supports the view that a specific concentration range of calcium is essential for outgrowth and motility and that calcium influences neuronal growth by an effect on actin structures within the growth cone. Using time-lapse video microscopy techniques, the effects of calcium ionophore A23187 on the rate of neurite outgrowth and growth cone motility in cultured embryonic mouse spinal cord neurons were examined. Concentrations in the range of 1-100 microM resulted in an inhibition of neuronal outgrowth, a loss of filopodia and a significant reduction in survival after seven hours. The results show that although the treatment with A23187 inhibited growth at all concentrations used, motility was inhibited only at the highest concentration indicating that the optimum calcium concentration for motility is higher than that for growth and that calcium mediates its effects on growth and motility at a very local level.

Animals↗
PubMed1997

Cerebral vasculitis--recognition, diagnosis and management.

Cerebral vasculitis is a serious but uncommon condition which presents considerable difficulties in recognition, diagnosis and treatment. We studied eight consecutive patients in whom this diagnosis was made. Despite the great diversity of symptoms and signs, we noted three clinical patterns: (i) acute or sub-acute encephalopathy, (ii) a picture with some similarities to multiple sclerosis ('MS-plus'), and (iii) features of a rapidly progressive space-occupying lesion. The identification of these patterns may help recognition of cerebral vasculitis. The diagnostic value of four investigative procedures not previously studied in cerebral vasculitis was assessed: ophthalmological examination using low-dose fluorescein angiography with slit-lamp video microscopy of the anterior segment (abnormal in 4/5 patients); spinal fluid oligoclonal band analysis (abnormal in 3/6 patients); anti-neutrophil cytoplasmic antibody assay (abnormal in 3/8 patients); and indium-labelled white-cell cerebral imaging (positive in only one patient). Treatment was with steroid alone (n = 2) or steroid with cyclophosphamide (n = 6). Seven patients responded clinically.

Adult↗
PubMed2003

Cadmium-induced mitochondrial membrane-potential dissipation does not necessarily require cytosolic oxidative stress: studies using rhodamine-123 fluorescence unquenching.

The impact of cadmium on the cellular redox state and mitochondrial membrane potential (psi(m)) has been studied by monitoring dichlorofluorescein (DCF), CMXRos (dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine), and Rh-123 fluorescence in 5-day-old TC7 cells, a highly differentiated clone of the human intestinal Caco-2 cell line. Flow cytometry analyses, using DCFH oxidation to DCF, clearly revealed that a 30-min incubation to 50 microM cadmium (Cd) is sufficient to induce reactive oxygen species (ROS) formation; this effect was completely eliminated by the presence of 50 mM mannitol for the 30-min incubation period, but mannitol only partially scavenged ROS for the longer period of time studied. Imaging studies using fluorescence video microscopy revealed a parallel increase in (DCF) fluorescence in the nuclear and cytoplasmic regions as soon as Cd was added to the exposure medium. Flow cytometry analyses monitoring CMXRos fluorescence clearly showed that Cd also leads to psi(m) disruption, but, contrary to what was observed for ROS formation, mannitol was completely inefficient in preventing this effect. Further investigation using fluorescence microscopy and Rh-123 fluorescence unquenching revealed that although mannitol did not protect against Cd-induced dissipation of psi(m), it considerably delayed the process. We found that Rh-123 unquenching, occurring during probe redistribution, is a suitable tool to monitor the decrease of psi(m). We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH(*), as well as the loss of psi(m). However, psi(m) dissipation does not necessarily require cellular OH(*) and may occur in the absence of apparent oxidative injury.

Caco-2 Cells↗
PubMed2003

Real-time observations of individual macaque sperm undergoing tight binding and the acrosome reaction on the zona pellucida.

Changes in binding affinity, acrosomal status, and motility of living sperm on the zona pellucida were for the first time in any mammalian species directly observed and analyzed with video microscopy. A single zona was air-dried and rehydrated on a microscope slide, and a coverslip supported by glass beads was added. Capacitated sperm were added together with Alexa-SBTI, a probe for acrosin that can detect the acrosome reaction. The heads of loosely attached sperm oscillated on the zona and the flagella beat symmetrically with a sigmoid-shaped waveform. Tight binding was observed after 16 sec as the sperm head became fixed in place on the zona. The shape of the flagellar beat simultaneously shifted to a more rigid, C-shaped waveform. The first signs of the acrosome reaction were detected within 11 sec of tight binding. Rapid flushing removed approximately 65% of sperm that were loosely attached but only 2% of those that were tightly bound. In the 2 min following the onset of tight binding, the lateral displacement of the flagellum increased by approximately 30% and the beat frequency decreased by 25%. Lignosulfonic acid (LSA) inhibited loose sperm attachment and the development of tight binding. LSA had no effect on the time of the acrosome reaction following tight binding or on changes in motility that followed tight binding. These data suggest that LSA affects the initial attachment or docking of sperm to the zona, a step that may align or recruit one or more specific zona receptors to be responsible for mediating the acrosome reaction.

Acrosome Reaction↗
PubMed1988

Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm.

The occurence and time course of capacitation, acrosomal loss, and hyperactivated motility require quantitative definition in order to characterize fertile human sperm. In this study, video microscopy and digital image analysis were used to measure curvilinear (VCL) and straight line (VSL) velocity, average linearity of progression (LIN [100 x VSL/VCL]), maximum and mean amplitude of lateral head displacement (ALH), beat-cross-frequency (BCF), DANCE (VCL x meanALH) and DANCEMEAN (meanALH/(LIN/100]. These parameters were measured for sperm in semen and in the swim-up fraction of washed cells during incubation for up to 24 h under in vitro fertilization (IVF) conditions. Acrosomal loss was monitored in the same population of washed cells by an immunofluorescence end-point assay. The greatest increase in mean values of motility parameters was observed when seminal sperm were washed free of seminal plasma. Increases continued for up to 6 h of incubation. Two subpopulations of hyperactivated sperm were identified; one type, not found in semen, showed star-spin trajectories, and constituted 3.0, 3.8, 4.5, and 4.1% of the swim-up population after 0, 3, 6 and 24 h of incubation. The second type, termed transitional showed a more progressive trajectory and constituted less than 1% in semen. In total, hyperactivated cells constituted 0.8% of cells in semen, 14.5% of the swim-up population with no incubation, and 23.1, 22.7, and 19.4% after 3, 6, and 24 h of incubation, respectively. Acrosomal loss in the swim-up population was delayed during the first 3 h of incubation, then increased from near 5% at 3 h to 7 and 12% at 6 and 24 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1988

Motility of rat spermatozoa at the site of fertilization.

This study was undertaken to examine the factors that may affect the numbers and motility patterns of spermatozoa at the site of fertilization. The contents of the oviductal ampullae of previously mated cycling or superovulated immature rats were examined microscopically. We determined whether spermatozoa were free or associated with cells and whether they exhibited hyperactivated motility, forward progressive motility, or were immotile. These data were correlated with the percentage of fertilized eggs. In addition, the beat pattern of hyperactivated spermatozoa was characterized by using high-speed video microscopy. At the time when half of the eggs were fertilized, ampullae of cycling rats contained an average of less than one motile spermatozoon per ampulla. Most of these motile spermatozoa were hyperactivated. About half of these were free in the ampulla and about half were in the cumulus or zona pellucida. Hyperactivated spermatozoa displayed a nonprogressive whiplash wave form with a high amplitude recovery stroke similar to that described in hamster and guinea pig spermatozoa capacitated in vitro. In addition to motile spermatozoa, we counted about three immotile spermatozoa for each motile spermatozoon. In superovulated, immature female rats, we found about ten times as many spermatozoa in each category as in cycling rats. From our observations, it is clear that very few spermatozoa reach the ampulla of the oviduct. Furthermore our observations suggest that in cycling rats progressively swimming spermatozoa may become hyperactivated shortly after entering the ampulla of the oviduct. They probably enter the cumulus mass within a short time or become immotile.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗