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At least 217 records · Page 12Linked to original sources
PubMed1998

Influence of thrombus components in mediating Staphylococcus aureus adhesion to polyurethane surfaces.

The role of protein and cellular components of thrombi in mediating bacterial adhesion on artificial surfaces was investigated in this study. The attachment of Staphylococcus aureus on polyurethane surfaces was observed directly using an automated video microscopy system. Surfaces were preconditioned with components of platelet-fibrin thrombi, including fibrinogen, thrombin, plasma, and isolated platelets. Experiments were performed in a radial flow chamber, and attachment rate constants were compared on the preconditioned surfaces in an effort to understand the complex relationship that exists between bacterial infection and thrombosis on synthetic biomaterials. Preadsorption of fibrinogen to surfaces significantly increased S. aureus adhesion compared to those preadsorbed with albumin alone while the presence of fibrin dramatically increased bacterial attachment compared to plasma preadsorbed surfaces. While the presence of adherent platelets also increased bacterial attachment, fibrin appeared to play a larger role in mediating bacterial adhesion on polyurethane surfaces. Striking results were obtained on the zwitterionic phosphonated polyurethane for a number of pretreatment conditions with regard to decreased bacterial adhesion and fibrinogen deposition.

Bacterial Adhesion↗
PubMed2000

Agonist-induced changes in cell shape during regulated secretion in rat pancreatic acini.

The actin cytoskeleton plays an important role in the mediation of exocytosis and the determination of cell shape. Experimentally induced changes in cell shape have been shown to affect stimulated secretion in pancreatic acini. In this study, we have examined whether physiologic agonists induce changes in acinar cell shape to modulate secretion. Computer-enhanced video microscopy, immunofluorescence confocal microscopy, and quantitative Western blotting were used to study cell shape changes and cytoskeletal dynamics in rat pancreatic acini. Amylase assays were performed to study the effect of the actin-myosin cytoskeletal antagonists latrunculin A, BDM, and ML-9 on secretion. We found that pancreatic acini underwent a prominent and reversible shape change in response to the physiologic secretory agonist cholecystokinin. This was accompanied by an apical activation of myosin II as well as a basolateral redistribution of both actin and myosin II. Cytoskeletal antagonists inhibited this shape change and attenuated stimulated amylase secretion. Therefore, in addition to acting as a barrier at the apex, the actin-myosin cytoskeleton may also function to modulate cell shape to further regulate stimulated secretion.

Actins↗
PubMed1998

Time course of the development of motor behaviors in the zebrafish embryo.

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.

Aging↗
PubMed1999

Cytotoxic T lymphocytes can induce a condemned state and synchronous post-mitotic apoptosis of daughter target cells.

We have used time-lapse video microscopy to study cytotoxic T lymphocyte (CTL)-mediated apoptosis of LDb fibroblast target cells at different phases of the cell cycle. When aphidicolin-synchronized target cells were exposed to the CTL clone F5, apoptosis occurred with similar morphology during G1, S/G2 and M phase, showing that apoptosis and mitosis are not mutually exclusive cellular events. Interestingly, following normal mitosis of target cells that had been previously contacted by CTL, pairs of daughter cells would occasionally undergo apoptosis within minutes of each other. Such synchronous post-mitotic apoptosis was also observed when using mitotically unsynchronized target cells, and also when using d11S T cell hybridomas as alternative Fas- (CD95-) based effector cells, even if these effectors were physically washed away after an initial period of co-incubation with the target cells. Our observations show that cytotoxic cells can induce a condemned state in pre-mitotic target cells, which can be inherited by both daughter cells, leading to their synchronous apoptosis after mitosis.

Animals↗
PubMed2001

Prolonged recycling of internalized neurotrophins in the nerve terminal.

BACKGROUND: Neurons require contact with their target tissue in order to survive and make correct connections. The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. However, the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body remains unclear. METHODS: Retrograde axonal transport was examined using the transport of I(125)-labeled neurotrophins from the eye to sympathetic and sensory ganglia. The phenomena was further studied by adding rhodamine-labeled nerve growth factor (NGF) to cultures of dissociated sympathetic ganglia and the movement of organelles followed with the aid of video microscopy. RESULTS: I(125)-labeled neurotrophins were transported from the eye to the sympathetic and sensory ganglia. A 100-fold excess of unlabeled neurotrophin, administered up to 4 h after the labeled material, completely prevented accumulation of labeled neurotrophin in the ganglia. The effect was specific for the labeled neurotrophin as administration of a high concentration of a different neurotrophin failed to inhibit the transport. In dissociated cultures, we found rapid binding of label, to surface membrane receptors, followed by an accumulation of labeled vesicles in the growth cone. Incubation of these cultures with unlabeled NGF led to a rapid loss of label in the growth cones. CONCLUSIONS: These results suggest that there is a pool of internalized neurotrophin, in vesicles in the nerve terminal, which is in rapid equilibrium with the external environment. It is from this pool that a small fraction of the neurotrophin-containing vesicles is targeted for retrograde transport. Potential models for this system are presented.

Animals↗
PubMed2000

Proliferative and migratory responses of astrocytes to in vitro injury.

An in vitro "scratch-wound" model was used to evoke and investigate some astroglial responses to mechanical injury. The changes in the morphology, locomotion, and proliferation of injured astrocytes were analysed under culture conditions devoid of blood-derived cells responsible for activating the inflammatory cascade. The rate of proliferation was determined by immunocytochemical detection of BrdU-incorporating cells located next to or far from the wound. The motility of individual cells and the mass-advancement of cell-assemblies were monitored by computer controlled video-microscopy both in injured monolayers and in preparations of single cells or aggregates of astrocytes. The large sets of digitalized data allowed a reliable statistical evaluation of changes in cell positions providing a quantitative approach for studies on dynamics of cell locomotion. The results indicated that cultivated astrocytes respond to injury (1) with enhanced nestin immunoreactivity at the expanding processes, (2) with increased mitotic activity exceeding the rate caused by the liberation from contact inhibition, but (3) without specific, injury-induced activation of cell locomotion. Some advantages and drawbacks of "scratch-wound" models of astrocytic responses to mechanical injury are presented and discussed.

Animals↗
PubMed2000

CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.

Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

Actins↗
PubMed2001

Cellular dynamics during evagination of the thyroid primordium in the chick embryo.

The thyroid forms as an outpouching of the ventral pharynx. Evidence supports the conclusion that formation of the thyroid pit is mediated by changes in the cytoskeleton that cause constriction of cell apices. However, it seems unlikely that a relatively flat epithelial sheet can be converted into a pit without either distortions of the surface or considerable rearrangement of cells to reduce surface area. Possible cellular rearrangements were investigated by tracing the movements of individual cells by using time-lapse video microscopy. Changes in shape of the primordium were investigated by marking with carbon and DiI and by scanning electron microscopy. Cell movements occurred only over short distances, mostly shifts relative to a neighbor, especially at the edge of the pit. Instead, cells rearranged into clusters that piled up at the edge of the pit and then tilted inside. Adjacent rings of pharyngeal cells were annexed by the growing thyroid, undergoing rearrangement into clusters, piling up at the edge, and moving inside the pit. The consequence was the formation of a series of shelf-like extensions within the cavity, representing successive generations of cell rings moving inside. These results have implications for the formation of other organs by evagination.

Animals↗
PubMed2005

Visualization and in situ analysis of leukocyte trafficking into the ankle joint in a systemic murine model of rheumatoid arthritis.

OBJECTIVE: To describe the kinetics of leukocyte migration into a distal joint during the development of chronic inflammation in a murine model of rheumatoid arthritis (RA), to identify leukocyte subpopulations recruited in the synovial vessels, and to test in real time the effects of an antiinflammatory compound on leukocyte-endothelial cell interactions in the arthritic joint. METHODS: We used intravital video microscopy (IVM), which was adapted to the microcirculation of the mouse ankle, to monitor the kinetics of leukocyte-endothelium interactions (rolling and firm adhesion) during the onset and progression of proteoglycan-induced arthritis (PGIA), a chronic autoimmune model of RA. Subpopulations of rolling and adherent leukocytes were identified by in vivo immunostaining. Leukocyte extravasation into the ankle joint was verified histologically. RESULTS: Between the onset of arthritis and the beginning of the destructive phase of PGIA, we found a steady increase in the number of leukocytes that exhibited firm adherence to the endothelium of synovial vessels, which clearly underscores the chronic, self-perpetuating character of joint inflammation in this autoimmune model. We showed, however, that granulocytes, and not T cells, constituted the major cell population that was continuously recruited to the inflamed ankle. Using IVM, we could detect instant changes in leukocyte adhesion behavior in the synovial vessels of the arthritic joint upon administration of a compound that antagonizes leukocyte rolling. CONCLUSION: IVM of the microcirculation of the mouse ankle could become an essential tool for investigating the mechanisms that regulate leukocyte migration to the joint in systemic models of RA as well as for preclinical testing of antiinflammatory therapies.

Animals↗
PubMed2002

Actin-based phagosome motility.

Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation.

Actins↗
PubMed2004

Early molecular events in the assembly of the focal adhesion-stress fiber complex during fibroblast spreading.

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed.

Actin Cytoskeleton↗
PubMed2006

Self-organized and highly ordered domain structures within swarms of Myxococcus xanthus.

Coordinated group movement (swarming) is a key aspect of Myxococcus xanthus' social behavior. Here we report observation of domain structures formed by multiple cells within large three-dimensional swarming groups grown on amorphous glass substrates, using the atomic force microscope (AFM). Novel analyses revealed that 90% of the wild type swarms displayed some form of preferential cell alignment. In contrast, cells with mutations in the social and adventurous motility systems displayed a distinct lack of cell alignment. Video microscopy observations of domain features of in vivo swarming M. xanthus cells were also consistent with the AFM data. The results presented here reveal that unique domain formation within swarms of wild type cells is a biologically driven process requiring the social and adventurous motility systems and is not a statistical phenomenon or thermodynamic process arising from liquid crystal behavior.

Microscopy, Atomic Force↗
PubMed2004

A model-based approach for automated in vitro cell tracking and chemotaxis analyses.

BACKGROUND: Chemotaxis may be studied in two main ways: 1) counting cells passing through an insert (e.g., using Boyden chambers), and 2) directly observing cell cultures (e.g., using Dunn chambers), both in response to stationary concentration gradients. This article promotes the use of Dunn chambers and in vitro cell-tracking, achieved by video microscopy coupled with automatic image analysis software, in order to extract quantitative and qualitative measurements characterizing the response of cells to a diffusible chemical agent. METHODS: Previously, we set up a videomicroscopy system coupled with image analysis software that was able to compute cell trajectories from in vitro cell cultures. In the present study, we are introducing a new software increasing the application field of this system to chemotaxis studies. This software is based on an adapted version of the active contour methodology, enabling each cell to be efficiently tracked for hours and resulting in detailed descriptions of individual cell trajectories. The major advantages of this method come from an improved robustness with respect to variability in cell morphologies between different cell lines and dynamical changes in cell shape during cell migration. Moreover, the software includes a very small number of parameters which do not require overly sensitive tuning. Finally, the running time of the software is very short, allowing improved possibilities in acquisition frequency and, consequently, improved descriptions of complex cell trajectories, i.e. trajectories including cell division and cell crossing. RESULTS: We validated this software on several artificial and real cell culture experiments in Dunn chambers also including comparisons with manual (human-controlled) analyses. CONCLUSIONS: We developed new software and data analysis tools for automated cell tracking which enable cell chemotaxis to be efficiently analyzed.

Cell Culture Techniques↗
PubMed2002

Multistep role for actin in initial closure of the mesencephalic neural groove in the chick embryo.

In a previous study, we have demonstrated that initial closure of the mesencephalic neural groove in the chick embryo is different from neurulation elsewhere. The neural groove invaginates, the walls appose and make contact in a ventrodorsal direction, and subsequently separate ventrally, forming an incipient neural tube lumen, which finally widens into a definitive lumen. In this study, a role for actin in the processes of this initial mesencephalic closure is studied. Based on rhodamine-phalloidin-stained sections, three distinct actin distribution patterns emerged, and time-lapse video microscopy revealed cytochalasin-D-reversible neurulation movements. We propose that actin is involved in formation and stabilization of the neural groove hinge point, in invagination of dorsal neuroepithelial cells into the neural groove, in the origin of the incipient lumen and the reinforcement of adhesion of the dorsal neural folds, and finally in the development of a wide lumen. Such a multifunctional effect of actin microfilaments within a narrow time window and at specific sites has not been reported yet.

Actins↗
PubMed2004

Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells.

The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.

Animals↗
PubMed1998

Carbon monoxide as a regulator of bile canalicular contractility in cultured rat hepatocytes.

This study aimed to examine the mechanism(s) by which carbon monoxide (CO), a product of heme oxygenase reaction, controls the contractility of bile canaliculus (BC) in hepatocytes. When BCs associated with the couplet cells in cultured rat hepatocyte suspension were observed using time-lapse video microscopy, they exhibited periodical contractions with a most-probable interval of 6 minutes under our experimental conditions. The addition of 1 micromol/L zinc protoporphyrin IX (ZnPP), a potent inhibitor of heme oxygenase, to the culture medium elicited a 40% shortening of the interval time together with an increase in intracellular calcium concentrations, while the same concentration of iron protoporphyrin IX did not induce such changes. The production of CO, which was 0.5 nmol/h/10(8) cells in the absence of ZnPP, diminished to less than 0.1 nmol/h/10(8) cells upon application of ZnPP. The ZnPP-elicited increases in both contractile frequency and intracellular calcium concentrations were attenuated by the addition of 1 micromol/L CO or 50 micromol/L 1,2-bis(2-aminophenoxy) ethane-tetraacetate, a calcium chelator. Clotrimazole or metyrapone, inhibitors of cytochrome P450-dependent monooxygenase activities, also attenuated the ZnPP-induced elevation of the contractile frequency. On the other hand, intracellular cyclic guanosine monophosphate (cGMP) contents were not altered significantly by the application of ZnPP or by CO. These results indicate that CO generated by heme oxygenase controls the BC function by changing intracellular calcium concentrations presumably through a mechanism involving the cytochrome P450 reaction.

Animals↗
PubMed2002

Time course of polyglutamine aggregate body formation and cell death: enhanced growth in nucleus and an interval for cell death.

Polyglutamine (polyQ) aggregate bodies are a hallmark of dentatorubral-pallidoluysian atrophy and related neurodegenerative disorders, although the relationship between aggregate body formation and cell death is not clear. We analyzed the kinetics of polyQ aggregate formation and the time intervals for cell death, tracking individual cells using fluorescence video microscopy, for the first time. Expanded polyQ tracts of atrophin-1 with or without nuclear localization signal (NLS) labeled with green fluorescent protein (GFP) were constructed, Q57NLS/GFP and Q56/GFP, respectively. All of the Q57NLS/GFP aggregate bodies were in nuclei, and all of the Q56/GFP aggregate bodies were in cytoplasm. Aggregates of Q56/GFP were larger than those of Q57NLS/GFP. Surprisingly, a kinetic analysis showed that the latter grew 5.37 times faster than the former. The time interval between transfection and cell death was shorter in Q57NLS/GFP, but the time between the end of the rapid growing phase of aggregation and the start of the cell death process did not show a significant difference. Aggregate growth was confirmed to correspond to the accumulated free polyQ by the time of starting aggregation. These findings suggest that aggregate body formation induced by expanded polyQ stretches is a self-limiting process and is enhanced by factor(s) in nuclei, whereas it is not tightly bound to the cell death process.

Animals↗
PubMed1995

Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum.

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement.

Animals↗