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K E Fogarty

Publications and source records attributed to K E Fogarty.

30 records · Page 2Linked to original sources
PubMed1991

Calcium gradients underlying polarization and chemotaxis of eosinophils.

The concentration of intracellular free calcium ([Ca2+]i) in polarized eosinophils was imaged during chemotaxis by monitoring fluorescence of the calcium-sensitive dye Fura-2 with a modified digital imaging microscope. Chemotactic stimuli caused [Ca2+]i to increase in a nonuniform manner that was related to cell activity. In cells moving persistently in one direction, [Ca2+]i was highest at the rear and lowest at the front of the cell. Before cells turned, [Ca2+]i transiently increased. The region of the cell that became the new leading edge had the lowest [Ca2+]i. These changes in [Ca2+]i provide a basis for understanding the organization and local activity of cytoskeletal proteins thought to underlie the directed migration of many cells.

Calcium↗
PubMed1991

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Animals↗
PubMed1989

Three-dimensional molecular distribution in single cells analysed using the digital imaging microscope.

Cellular changes in molecular distribution are believed to underly a wide range of cell functions. In order to investigate changes in molecular distribution in single cells utilizing fluorescent probes we have developed a digital imaging microscope. The system, consisting of both hardware and software, automatically acquires 3-D data sets consisting of optical sections and then processes such data to facilitate the analysis of molecular distribution in single cells. The first major step in processing reverses distortion introduced principally by the optics of the fluorescent microscope. Various procedures for accomplishing this task are compared and a method based on regularization theory is shown to give superior results for several different 3-D images. Following this step features of interest are automatically extracted from 3-D images utilizing an artificial 3-D visual system. This artificial visual system utilize a system of spatial filters to identify regional characteristics of images, the information obtained from these filters being used to identify and characterize clusters of molecules within the image. This information is then utilized to construct a 3-D graphical model of molecular distribution in single cells. Such models are displayed in 3-D and may further analysed utilizing interactive 3-D computer graphics. These methods are illustrated by results obtained regarding alpha-actinin distribution in single smooth muscle cells.

Actinin↗
PubMed1986

Development and application of a three-dimensional artificial visual system.

A three-dimensional artificial visual system has been developed to aid in the analysis of 3-D fluorescence images of smooth muscle cells. The system consists of three sets of 3-D spatial filters that decompose the image to enable a simple recombination algorithm to locate the discrete bodies of protein concentration in a cell, classify the concentration bodies as globular or oval, and determine the 3-D orientation of the oval bodies. A graphic model of the protein concentration is created from the data provided by the artificial visual system. Patterns of organization in the distribution of the protein bodies are investigated using an interactive graphics system.

Animals↗
PubMed1985

Computerized analysis of TV images for ultrasensitive monitoring of the reaction of fluorochrome with protein.

Preparation of protein-specific fluorescent probes with the desired degree of fluorochrome can be greatly facilitated by a technique that combines thin-layer chromatography (TLC) with quantitative image analysis (QIA). Using TLC/QIA, the investigator can determine the fluorochrome/protein ratio on-line with only a few micrograms of protein as fluorochrome is conjugated to protein. In addition, this technique allows rapid quantitation of dye noncovalently adsorbed to fluorochrome-labeled protein.

Chemical Phenomena↗
PubMed1983

Distribution of alpha-actinin in single isolated smooth muscle cells.

In order to probe the organization of the contractile machinery in smooth muscle, we have studied the distribution of alpha-actinin, a protein present in high concentration in dense bodies, structures apparently analogous to the Z-disks of striated muscle. Localization of alpha-actinin in single isolated smooth muscle cells of the stomach muscularis of Bufo marinus was determined by analysis of the pattern of anti-alpha-actinin staining in single fluorescence photomicrographs, stereo pair micrographs, and computerized three-dimensional reconstructions from multiple image planes. The distribution of anti-alpha-actinin and antitubulin staining was compared in contracted and relaxed cells. The studies revealed that alpha-actinin is present in high concentrations in fusiform elements (mean axial ratio = 4.82) throughout the cytoplasm and in larger, more irregularly shaped plaques along the cell margins. Many of the fusiform-stained elements are organized into stringlike arrays characterized by a regular repeating pattern (mean center-to-center interspace = 2.2 +/- 0.1 micron). These linear arrays appear to terminate at the anti-alpha-actinin stained larger plaques along the cell margin; several of these strings often run in parallel with their elements in lateral register. While this general pattern of organization is maintained in cells during contraction, the distance between successive stained elements in stringlike arrays is decreased. We suggest that the decrease in the distance between elements in these strings results from shortening of materials that constitute these linear arrays. We do not believe that the shortening within these arrays reflects compression by forces generated elsewhere within the cell, as the reorganization of noncontractile microtubules is qualitatively different from the changes in the pattern of anti-alpha-actinin staining.

Actinin↗
PubMed1978

Segmental reflexes mediated by joint afferent neurons in cat knee.

1. Reflex effects mediated by knee joint afferent neurons were tested using the electrically evoked monosynaptic reflex in motoneurons innervating muscles which act about the knee. 2. Joint afferents were excited using small extension movements of the knee, stimuli which have been shown to be effective in activating a large fraction of the myelinated fibers in joint nerves. Because of the variability of reflex responses observed by others under similar experimental conditions, experimental control and data collection were done automatically under computer control. Large numbers of trials were presented in random sequence, and the resulting data were sorted and averaged. 3. Positive feedback was observed in both knee extensor (vasti) and knee flexor (posterior biceps-semitendinosus (PB-ST)) motoneuron populations. Vasti motoneurons were excited, and PB-ST motoneurons were inhibited by knee extensions when the posterior articular nerve (PAN) was intact; these effects were lost or substantially reduced when the PAN was cut. 4. Using knee extension stimuli, no reliable reflex effects were observed which could be attributed to afferents in medial articular nerve.

Animals↗
PubMedDate not supplied

Ca2+ imaging in single living cells: theoretical and practical issues.

The measurement of intracellular calcium ion concentrations [( Ca2+]i) in single living cells using quantitative fluorescence microscopy draws from a diverse set of disciplines, including cellular biology, optical physics, statistics and computer science. Over the last few years, we have devised and built a number of systems for measuring [Ca2+]i with Fura-2, and have applied them in the exploration of a wide range of biological processes controlled by Ca2+. In this report we discuss these systems and their advantages and limitations. We also describe the theoretical and practical problems associated with using Fura-2 to measure [Ca2+]i, and the solutions that we, and others, have developed to overcome them. The approaches described should provide useful guidance for others interested in imaging [Ca2+] distribution in living cells. The factors that limit current methods are discussed, and areas for future development are highlighted.

Animals↗
PubMedDate not supplied

Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2.

Calcium is believed to control a variety of cellular processes, often with a high degree of spatial and temporal precision. For a cell to use Ca2+ in this manner, mechanisms must exist for controlling the ion in a localized fashion. We have now gained insight into such mechanisms from studies which measured Ca2+ in single living cells with high resolution using a digital imaging microscope and the highly fluorescent Ca2+-sensitive dye, Fura-2. Levels of Ca2+ in the cytoplasm, nucleus and sarcoplasmic reticulum (SR) are clearly different. Free [Ca2+] in the nucleus and SR was greater than in the cytoplasm and these gradients were abolished by Ca2+ ionophores. When external Ca2+ was raised above normal in the absence of ionophores, free cytoplasmic Ca2+ increased but nuclear Ca2+ did not. Thus, nuclear [Ca2+] appears to be regulated independently of cytoplasmic [Ca2+] by gating mechanisms in the nuclear envelope. The observed regulation of intranuclear Ca2+ in these contractile cells may thus be seen as a way to prevent fluctuation in Ca2+-linked nuclear processes during the rise in cytoplasmic [Ca2+] which triggers contraction. The approach described here offers the opportunity of following changes in Ca2+ in cellular compartments in response to a wide range of stimuli, allowing new insights into the role of local changes in Ca2+ in the regulation of cell function.

Animals↗