Observations of exocytosis in fucus vesiculosus gametes using video-enhanced light microscopy: a video report.
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Eukaryota↗
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Dendroaxonal transcytosis of transferrin in cultured hippocampal and sympathetic neurons.
Previous studies using overexpressed polymeric immunoglobulin receptor in cultured neurons have suggested that these cells may use a dendroaxonal transcytotic pathway (Ikonen et al., 1993; de Hoop et al., 1995). By using a combination of semiquantitative light microscopy, video microscopy, and a biochemical assay, we show that this pathway is used by the endogenous ligand transferrin (Tf) and its receptor. Labeled Tf added to fully mature hippocampal neurons changes the intracellular distribution of its receptor from preferentially dendritic shortly after addition to dendritic and axonal at longer times. Incubation of living neurons with (caged)FITC-Tf followed by uncaging in the dendrites results in the later appearance of fluorescence in the axon of the same cell. In "chambered" sympathetic neurons in culture, 125I-Tf or iron as 55Fe-Tf added to the cell body/dendrite chamber is recovered in the axonal chamber, showing that internalized ligand from the cell body-dendrite area is released at the axonal end. Finally, we show that excitatory neurotransmitters increase Tf receptor transcytosis, whereas inhibitory neurotransmitters reduce it. The dendritic uptake, transcytotic transport, and axonal release of physiologically active Tf demonstrated here could be envisioned for other trophic factors and therefore have important consequences for neuronal anterograde target maturation. Moreover, the changes in transcytosis after neurotransmitter addition may be important in the cellular responses that follow electrical activation.
Animals↗
Dynamic measurements of intracellular aminopeptidase activity in hepatocytes using multiparameter digitized video fluorescent microscopy.
Digitized video fluorescent microscopy (DVFM) is a powerful technique for quantitating multiple processes in living cells. However, techniques for measuring protease activity by DVFM are not available. Our aim was to develop an approach for measuring aminopeptidase activity using DVFM. We conjugated glycine-7-amino-4-methylcoumarin-3-acetic acid (glycine-AMC-3-acetic acid) to dextran using a PEG bridge. Glycine-AMC-3-acetic acid-PEG-dextran was microinjected into cultured rat hepatocytes along with rhodamine-dextran. Glycine-AMC-3-acetic acid-PEG-dextran is nonfluorescent, but aminopeptidase hydrolysis of the glycine-AMC bond liberates the fluorescent AMC-3-acetic acid-PEG-dextran within the cell. Following microinjection, rhodamine-dextran fluorescence remained constant while AMC-3-acetic acid-PEG-dextran fluorescence increased in a linear fashion over time reflecting proteolytic cleavage of the glycine-AMC bond. AMC-3-acetic acid-PEG-dextran and rhodamine-dextran fluorescence were cytosolic as evidenced by diffuse fluorescence and colocalized. Because rhodamine-dextran fluorescence remained constant and the probes colocalized, the fluorescent ratio of AMC-3-acetic acid-PEG-dextran/rhodamine-dextran could be used to measure proteolysis. Basal rates of proteolysis were 9 +/- 3 ratio units/10 min. Comicroinjection of the aminopeptidase inhibitor, bestatin, along with the dextran probes abolished proteolysis. Addition of the calcium ionophore, 4-Br-A23187, increased proteolysis 12-fold to 107 +/- 14/10 min (P < 0.01). We have developed a novel, dynamic technique for measuring pH-sensitive, Ca(2+)-dependent aminopeptidase activity in living cells using DVFM. This approach may be used for the measurement of other peptidase activities by synthesizing peptidase-specific peptidyl-AMC-3-acetic acid-PEG-dextran conjugates.
Aminopeptidases↗
Molecular dissection of the fibroblast-traction machinery.
Motile fibroblasts generate forces that can be expressed as cell migration or as traction, the drawing-in of extracellular matrix. Traction by cultured fibroblasts can induce a rapid concerted reorganization of collagen gel, creating a pattern of collagen alignment similar to that seen in tendons and ligaments. In such fibrous connective tissues, after pattern morphogenesis is complete, ongoing traction may be responsible for the maintenance of proper form and function. The molecules that generate and transmit forces have been catalogued; however, how these nanometer-scale molecules contribute to millimeter-scale patterns has not been directly tested. Here, we placed pairs of explants of human periodontal ligament fibroblasts in collagen gels, where ligament-like straps of anisotropic collagen formed on the axes between them. We scrutinized the traction apparatus using electron microscopy, video microscopy, and computer-based pattern analysis, augmented with pharmacologic inhibitors of cytoskeletal function. Patterning was marked by the co-alignment of collagen, fibroblasts, and their actin cytoskeletons, all parallel to the axis between explants. The pattern was diminished by depolymerizing actin filaments or by blocking myosin activity, but was accentuated by depolymerizing microtubules. The plasma membrane also seems to contribute to the traction force. These molecular components combine to exert a sub-maximal traction force on the matrix, suggesting that the force may be regulated to ensure tissue tensional homeostasis.
Actin Cytoskeleton↗
Actin based motility on retraction fibers in mitotic PtK2 cells.
When PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase-dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 microns/minute. As they move in they increase progressively in phase-density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed.
Actins↗
Distribution of hematoporphyrin derivative in the rat 9l gliosarcoma brain tumor analyzed by digital video fluorescence microscopy.
A digital video fluorescence microscopy technique was used to evaluate the distribution of hematoporphyrin derivative (HPD) in the rat intracerebral 9L gliosarcoma brain-tumor model at 4, 24, 48, and 72 hours after intravenous administration of 10 mg/kg of the drug. Compared to surrounding normal brain, there was significant preferential uptake of HPD into the tumor. In sections surveyed, fluorescence reached a maximum value by 24 hours; however, only 33% to 44% of the tumor was fluorescent. In contrast, fluorescence within the surrounding normal brain was maximum at 4 hours, but was present in less than 1% of the brain tissue evaluated. The effect of HPD sensitization to a laser light dose (633 nm) of 30 joules/sq cm delivered through the intact skull was evaluated histologically in 10 rats. A patchy coagulation necrosis, possibly corresponding to the distribution of HPD fluorescence seen within the tumor, was observed. There was evidence that photoradiation therapy (PRT) affects defective tumor vasculature and that a direct tumor cell toxicity spared normal brain tissue. Despite these findings, limited uptake of HPD in tumor and the brain adjacent to tumor may decrease the effectiveness of PRT in the 9L gliosarcoma brain-tumor model. Because of the similarity between the capillary system of the 9L tumor and human brain tumors, PRT may have a limited therapeutic effect in patients with malignant brain tumors.
Animals↗
Effects of heat shock on the functional morphology of cell organelles observed by video-enhanced microscopy.
In living astrocytes and MDCK cells we observed morphological phenomena during and after heat shock (HS) utilizing our new perfusable microchamber system, which monitors pH, pO(2), pCO(2), and temperature. By means of electronic light microscopy and confocal laser scanning microscopy, mitochondria were demonstrated to swell and to reduce their motility. The specific fluorescent probe MitoTracker Green revealed that the mitochondrial morphology changed from a rodlike into an annular shape with a central vacuole-findings which were corroborated by transmission electron microscopy. After HS (shift from 37 degrees C to 45 degrees C for 15 min) the mitochondrial membrane potential (DeltaPsi(m)) was depressed in most but not all mitochondria as monitored with the fluorescent probe JC-1. The dual emission images of JC-1 illustrated a heterogeneous red staining of distinct areas of single mitochondria. The shape changes as well as the drop of the membrane potential of the mitochondria indicated severe cellular stress and a direct intervention on the mitochondrial permeability transition.
Animals↗
Video-enhanced microscopy for analysis of cytoskeleton structure and function.
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Animals↗
["Spatial shift alignment (SSA)"--a new method for determining blood flow velocity in video capillary microscopy].
A new technique (spatial shift alignment [SSA]) for measuring capillary blood velocity in human capillaries in the skin is described. A capillaroscopic image is digitized by means of a frame grabber. Within the lumen of a suitable capillary limb a region of interest known as a slot is placed. At a frame frequency of 25 per second, this slot is copied in a free part of the frame grabber store along a horizontal axis. A static space-time image with light diagonal bands representing the plasma gaps in their passage through the capillary limb is obtained. By determining the gradient of the diagonal bands, a measure of the capillary blood velocity is obtained. The SSA system was validated by means of a simulated model using known fixed velocities between 0 and 1.0 mm/sec (r = 0.979, n = 24). The procedure can be employed with any image processing systems, and enables continuous monitoring of the velocity measurements. Since it is time-saving and just as accurate as the frame-to-frame method, SSA could be widely applied to the measurement of blood cell velocity in capillaroscopy.
Blood Flow Velocity↗
[Video capillary microscopy studies of probands without and with occlusions of the finger arteries before and following hyper- and isovolemic hemodilution].
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Arterial Occlusive Diseases↗
[Clinical video-capillary microscopy].
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Blood Flow Velocity↗
Asymmetric illumination contrast: a method of image formation for video light microscopy.
Images with high resolution and exceptionally broad gray scale can be obtained by the application of video contrast enhancement to an optimized procedure for imaging transparent objects with oblique rays of illumination. This technique is simple to set up. A conventional microscope with a light source whose position can be adjusted and a video camera with controls for gain and black level are the only essential components. Features such as high resolution, optical sectioning, control of contrast, and operation under low light intensity make this technique preferable, in several instances, to currently used video microscopy techniques.
Microscopy↗
Exocytosis in living salivary glands: direct visualization by video-enhanced microscopy and confocal laser microscopy.
Although exocytosis is widely believed to involve granule movement, membrane fusion and the emptying of granule content, direct study of these processes has been difficult in living cells because of the limited resolution of conventional light microscopy. Using video-enhanced microscopy and confocal laser microscopy, we have now studied these processes in living rat parotid and submandibular gland acinar cells. Under a differential interference contrast (DIC) microscope equipped with a CCD camera and a high speed image processor, secretory granules were in general stationary even after secretory stimulation with isoproterenol (IPR). Following IPR stimulation, however, there were abrupt changes in light intensity of secretory granules, and many granules disappeared. Confocal microscopy was then performed to confirm whether the observed changes in granules were related to membrane fusion and content release. For this, cells were perfused with the fluid-phase tracer Lucifer Yellow; confocal images thus obtained clearly demonstrated the appearance of fluorescence in omega-shaped invaginations of the apical plasma membrane which corresponded to the sites at which changes were observed in DIC images. The time sequence analyses of confocal images showed that there was a repetitive appearance and disappearance of omega-shaped fluorescent foci at the apical plasma membrane until most of the granules were depleted. During this time, there did not appear to be any significant expansion of the apical plasma membrane and if endocytic uptake of the tracer occurred, it was below the limit of detection. These observations provide new insights into the exocytotic process in salivary glands and are at variance in some respects with previous interpretations made from electron microscopy.
Animals↗
Video fluorescence microscopy as a tool for the study of cellular heterogeneity in epithelia.
The use of functional fluorescent dyes has allowed us to monitor intracellular pH in individually identified cells in renal epithelia. Using video microscopy we simultaneously measured the change in intracellular pH in several contiguous cells in response to various maneuvers. The video equipment included a silicon intensified target camera, a VHS videocassette recorder, a high resolution monochrome monitor, a video photometric analyzer and a 2-channel chart recorder. This equipment had a spatial resolution of 1 micron by light microscopy and a response time of less than 200 ms; it allowed us to perform double fluorescent labeling and obtain reliable measurements of intracellular pH, independent of gain, regardless of the location of the image on the screen. Using this video system we have shown that there is substantial heterogeneity in activity of H+/HCO3- transport pathways among adjacent cells in a monolayer of cells cultured from the rat renal inner medullary collecting duct. In isolated perfused rabbit renal cortical collecting ducts, video microscopy allowed us to show that there are two different types of intercalated cells: one that exhibits apical Cl-/HCO3- exchange and one that does not. Both show alkaline intracellular pH with respect to non-acid-base transporting epithelia. Video microscopy has several advantages over conventional microspectrophotometry. It provides rapid data acquisition along with increased sensitivity and the capacity for some subcellular analyses. One is able to analyze several individually identified cells during an experimental maneuver. The present video system was assembled for less than $15,000 and permits a more complete analysis of an epithelium than either single-cell photometry or spectrophotometric analysis of thousands of cells in suspension or monolayers.
Animals↗
Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia.
Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia. Experimental Parasitology 97, 9-14. Giardia infection is predominant in the small intestine of vertebrates, where the trophozoites attach to epithelial cells and adversely affect the microvilli and other epithelial cell structures. Giardiasis, the disease caused by this protozoan, is very common in developing countries and mainly affects children. Drugs currently used to treat Giardia infection, such as some benzimidazole derivatives, were originally designed to treat helminthic infections. Many of the drugs are known to cause severe side effects and disturbances to the patient. Using transmission electron microscopy and video-light microscopy, we studied the effects of pyrantel pamoate, a drug commonly used in the treatment of helminthic infections in horses and ruminants, on Giardia lamblia trophozoites. Pyrantel pamoate was administered to Giardia cells in four different concentrations. Using video-light microscopy, we observed the decrease in flagella beating frequency and severe changes in the lateral flange and in the general aspect of the cell. Using transmission electron microscopy, we observed changes in the cytoplasm and peripheral vesicles. The flagella and adhesive disk structure were not affected. Apparently, the effects of pyrantel pamoate are irreversible.
Animals↗
Video-enhanced microscopy of organelle movement in an intact epithelium.
Digitally enhanced video microscopy has provided improved optical resolution in the study of intracellular organelle/particle movement, particularly in extruded axoplasm and certain thin single cell systems. We report here, for the first time, particle movement in an intact, isolated epithelium, the killifish proximal convoluted tubule. Cytoplasmic particles exhibited predominantly unidirectional linear movement approaching several microns in length, sometimes with multiple turns. The velocities of 34 particles measured in 11 cells averaged 0.29 microns/sec (range, 0.007-3.1 microns/sec). Microtubules--the well-established basis for organelle movement in cells--were present but were sparsely represented in electron micrographs of these cells. Video-enhanced microscopic techniques can now be applied to the study of organelle/particle movement in an intact epithelium.
Animals↗
Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy.
Video cameras with contrast and black level controls can yield polarized light and differential interference contrast microscope images with unprecedented image quality, resolution, and recording speed. The theoretical basis and practical aspects of video polarization and differential interference contrast microscopy are discussed and several applications in cell biology are illustrated. These include: birefringence of cortical structures and beating cilia in Stentor, birefringence of rotating flagella on a single bacterium, growth and morphogenesis of echinoderm skeletal spicules in culture, ciliary and electrical activity in a balancing organ of a nudibranch snail, and acrosomal reaction in activated sperm.
Acrosome↗
Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.
We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.
Animals↗