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At least 775 records · Page 43Linked to original sources
PubMed1988

Microtubule-associated organelle and vesicle transport in fibroblasts.

Allen Video-enhanced contrast/differential interference contrast (AVEC-DIC) microscopy was used in conjunction with video intensification immunofluorescence microscopy to demonstrate that organelles and vesicle (particles) can move in either direction along microtubular linear elements in fibroblasts [Hayden et al., 1983]. Since it is not possible to determine the number of microtubules making up a linear element with light microscopy alone, AVEC-DIC microscopy was used in conjunction with whole-mount electron microscopy to show bidirectional transport along a single microtubule [Hayden and Allen, 1984]. These studies demonstrate that the structural polarity of the microtubule does not determine the direction of particle motion, and since dynein is an asymetric molecule, a simple microtubule-dynein-particle hypothesis cannot explain bidirectional transport along a single microtubule. Very little is known about regulation of particle transport in most cell types. Human embryonic lung fibroblasts grown on glass coverslips were serum-deprived for 24 hours and re-fed with serumless medium; the particle translocations/5 minutes were then determined. The cells were then re-fed with either serumless medium, serum-containing medium, or serumless medium containing some bioactive factor, and the particle translocations/5 minutes were again determined for the same cells. Medium containing 10% fetal bovine serum inhibited particle translocation by 51.8%. Of the bioactive factors tested, only vasopressin produced a significant reduction in particle translocations (38%). This suggests that protein kinase C or calcium/calmodulin kinase could be involved in regulating particle transport.

Cells, Cultured↗
PubMed1996

Embryonic Xenopus neurites integrate and respond to simultaneous electrical and adhesive guidance cues.

Nerve cells detect and respond to multiple extrinsic guidance cues during development and regeneration using a motile growth cone. Navigational decisions may be required of the growth cone when different guidance cues are encountered simultaneously. We have tested the relative potencies of two opposing cues by presenting Xenopus spinal cord nerve cells growing on a micropatterned laminin culture substratum with an orthogonal DC electric field. Substrata composed of repeating 25-micron laminin tracks and spaces failed to influence the position of neuritogenesis from nerve cell soma. Once established, however, growth cone movement was constrained by laminin tracks such that neurites of 65% of cells were aligned after 5 h in vitro. Two hours after the application of a 100-140 mV/mm DC field the majority of cells remained aligned with the laminin tracks. Around 70% of Xenopus neurites normally orient cathodally on homogenous laminin substrata; therefore the galvanotropic response was impeded by prior exposure to a patterned laminin substrate. However, a proportion of aligned neurites did orient cathodally and evidence of a response to both directional cues was even found within the same cell. Video-enhanced contrast, differential interference contrast (VEC-DIC) microscopy was used to examine the detailed behavior of growth cones on micropatterned laminin substrata. The present study has demonstrated that growth cones can detect and integrate at least two morphogenetic guidance cues simultaneously. The strength of the galvanotropic response in Xenopus growth cones, however, was often insufficient to override established adhesive guidance in this model system.

Animals↗
PubMed1993

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells.

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using HEp-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the HEp-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100 degrees C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of HEp-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to HEp-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.

Bacterial Adhesion↗
PubMed1995

Non-amoeboid locomotion of cultured microglia obtained from newborn rat brain.

The movements of cultured microglia obtained from newborn rat brain were examined by video enhanced-differential interference contrast (VEC-DIC) microscopy. Active microglia measured 9.03 +/- 1.06 microns in diameter (mean +/- SD, n = 33; range, 7.03-10.36 microns). The microglia appeared to become smaller with spread of lamellipodia. The short axis of actively moving microglia measured 7.03 +/- 0.49 microns (n = 7). The lamellipodia were thin, transparent and developed rapidly around the cell body (maximal speed of extension, 4 or 5 microns/s). When shear stress from the medium was applied to the surface of cultured cells, the microglia swam with flat lamellipodia serving as sails in the stream. Spontaneous non-amoeboid movements of microglia were observed: they pivoted, circled and marched in various directions using their lamellipodia. The angular speed of rotation was maximally 3 degrees/s. In 5 marching cells, the average speed (distance/s) was calculated at 1.01 +/- 0.54 microns/s (ca. 60 microns/min or 3.6 microns/h).

Animals↗
PubMed1992

Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth.

Our study explores the mechanisms behind neurite galvanotropism. Using phase, differential interference contrast and ratiometric fluorescence microscopy, we reveal four responses of N1E-115 mouse neuroblastoma cells to 0.1-1.0 mV/microns uniform DC electric fields: cathode-directed neurite initiation and elongation, cathode-biased growth cone filopodial protrusions, transient cathode-localized calcium increases, and persistent cathode-localized membrane depolarizations. These newly demonstrated events are temporally and spatially correlated, suggesting that they are causally related. The calcium increases are prevented by calcium channel blockers and by the removal of extracellular calcium. We therefore propose that the observed field-induced membrane depolarizations activate voltage-dependent calcium channels, resulting in cathode-localized calcium influx. This, in turn, may initiate the observed cathode-biased growth cone filopodial protrusions, followed by the cathode-directed neurite elongation.

Animals↗
PubMed1996

Imaging exocytosis and endocytosis.

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.

Animals↗
PubMed2002

Observation of topical catalysis by sphingomyelinase coupled to microspheres.

Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner.

Catalysis↗
PubMed2000

Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans.

The capsular swelling or quellung reaction was reported almost 100 years ago and described the effect of Abs on the appearance of microbial capsules. Despite widespread use to assess Ab binding to capsules, relatively little is known as to the mechanism of this effect or its biological consequences. The fungus Cryptococcus neoformans is an attractive system to study capsule reactions because it has a large polysaccharide capsule that is readily visible by light microscopy. When viewed by differential interference contrast microscopy, binding of mAb to C. neoformans cells produced two distinct capsular reactions that depended on the Ab epitope specificity and the yeast serotype. In the first pattern, termed "rim," the capsule appears transparent with a highly refractive outer edge. In the second pattern, termed "puffy," the capsule appears opaque and lacks a highly refractive outer rim. mAbs that bind with a rim pattern suppress the overall rate of C3 deposition on the yeast via the classical and alternative complement pathways. In contrast, mAbs that bind with a puffy pattern do not affect C3 deposition. Protective and nonprotective IgM mAbs produce rim and puffy patterns, respectively. These results indicate that: 1) capsule reactions are a consequence of Ab-induced changes in capsular refractive index; 2) the type of capsule reaction depends on the Ab specificity; and 3) Ab-induced changes in refractive index correlate with biological activities important for host defense against C. neoformans. Our results provide the first evidence associating distinct capsule reaction patterns with Ab biological activity.

Antibodies, Monoclonal↗
PubMed2005

Study on cyclospora cayetanensis associated with diarrheal disease in Nepal and Loa PDR.

A study of diarrheal diseases associated with Cyclospora cayetanensis was conducted in Nepal and Lao PDR. A total of 2083 samples were included in this study. Samples in Nepal were collected from October 1999 to August 2002 whereas samples in Lao PDR were collected from February 2002 to June 2003. C. cayetanensis was detected by direct microscopy using ultraviolet and differential interference contrast microscopy. The overall positive rate in Nepal was 9.2% (128/ 1397). A higher positive rate was observed in children aged 10 years and under (11.1%) and was lowest in the age group of 51-60 years (3.1%). A significantly higher positive rate was observed in the summer (rainy season) (12.6%) with the lowest prevalence in the spring (dry season) (1.8%) (p < 0.05). The positive rate was closely associated with rainfall (ml/month). Interestingly, only one of the total 686 samples (0.1%) from Lao PDR was found to be positive for Cyclospora oocysts.

Adolescent↗
PubMed1988

Micropruning: the mechanism of turning of Aplysia growth cones at substrate borders in vitro.

Growth cones of Aplysia californica neurons were observed with video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy as they turned at a border between poly-L-lysine-treated and untreated glass. Growth cones that turned generally developed 2 distinct active areas of filopodial and veil formation, much in the way of growth cones undergoing branching. Both active areas advanced, but turning of the neurite occurred through the selective resorption of the incipient branches developing on the untreated substrate. Thus, micropruning of developing regions of the growth cone, rather than the asymmetric extension of filopodia or veils, was primarily responsible for directing neurite growth. We present the hypothesis that abrupt turns by growing neurites are mediated by 2 sets of signals, one causing growth cone splitting, and a second set regulating the survival of the separate branches.

Animals↗
PubMed1979

Cell-to-substratum contacts in living cells: a direct correlation between interference-reflexion and indirect-immunofluorescence microscopy using antibodies against actin and alpha-actinin.

Rat mammary cells growing on glass coverslips were photographed first using interference-reflexion microscopy and then after processing for indirect-immunofluorescence microscopy with antibodies to actin or to alpha-actinin. A comparison of the images of the same cell given by the 2 microscopical procedures indicates that the focal contacts between the cell and the substratum correspond to distal ends of microfilament bundles, and the these bundles are only in limited areas close to the substratum. The focal contracts are rich in alpha-actinin which has been proposed as a membrane-anchorage protein for microfilament bundles. Use of stereo immunofluorescence microscopy allows a direct comparison between the interference-reflexion image, and the underside of the cell after staining with antibodies to actin or alpha-actinin.

Actinin↗
PubMed1999

Keratocytes generate traction forces in two phases.

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.

Actins↗
PubMed1994

Cytoskeletal movements and substrate interactions during initiation of neurite outgrowth by sympathetic neurons in vitro.

The initial outgrowth of neurites from chick sympathetic neurons grown in vitro was investigated by time-lapse microscopy with laser-scanning and conventional light microscopes. Video-enhanced contrast, differential interference contrast optics (VECDIC) were used to monitor movements of neuronal cytoplasm, as well as the movements of small beads attached to the surface membrane, and interference reflection microscopy (IRM) was used to determine the concomitant pattern of attachment to the growth substrate (polyornithine or laminin). Related changes in the distributions of actin filaments, microtubules, and neurofilaments were determined by fluorescence labeling methods. Neurite formation on both substrates entailed invasion of the actin cores of filopodia by cytoplasm containing microtubules and neurofilaments. Small beads attached to the surface membrane surrounding the cytoplasm moved outward simultaneously with the cytoplasm. Cytoplasm invaded filopodia of neurons plated on laminin soon after attachment to the substrate or, for neurons generated in vitro, within as little as 3 min after cytokinesis. However, cytoplasm invaded filopodia of neurons grown on polyornithine only when they contacted a three-dimensional object such as another cell or a large, polyornithine-coated polystyrene bead. The observation that adhesion of filopodia to polyornithine-coated beads can initiate neurite formation is inconsistent with the commonly held view that neurite formation requires adhesion mediated by specific cell adhesion molecules. Simultaneous IRM and DIC imaging showed that cytoplasm invaded filopodia when only their tips were closely apposed to a substrate but not when they were closely apposed to a substrate along their entire lengths. These findings help to elucidate the mechanisms by which interactions between the cytoskeleton and the growth substrate initiate and produce the neuronal movements that lead to the formation of neurites.

Actins↗
PubMed2006

Effective elimination of laser interference fringing in fluorescence microscopy by spinning azimuthal incidence angle.

Laser illumination used in both conventional widefield epi-fluorescence as well as in total internal reflection fluorescence (TIRF) microscopy is subject to nonuniformities in intensity that obscure true image details. These intensity variations are interference fringes arising from coherent light scattering and diffraction at every surface in the laser light's optical path, including the lenses, mirrors, and coverslip. We present an inexpensive technique for effectively eliminating these interference fringes based upon introduction of the excitation laser beam by oblique through-the-objective incidence coupled with rapid azimuthal rotation of the plane of incidence. Although this rotation can be accomplished in several ways, a particularly simple method applicable to a free laser beam is to use an optical wedge, spun on a motor, which diverts the beam into a hollow cone of fixed angle. A system of lenses converts this collimated beam cone into a focused spot that traces a circle at the objective's back focal plane. Consequently, a collimated beam with fixed polar angle and spinning azimuthal angle illuminates the sample. If the wedge is spun rapidly, then the different interference patterns at every particular azimuthal incidence angle average out over a single camera exposure to produce an effectively uniform field of illumination.

Algorithms↗