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F S Fay

Publications and source records attributed to F S Fay.

At least 55 records · Page 3Linked to original sources
PubMed1993

A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus.

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

Cell Nucleus↗
PubMed1993

Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria.

Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.

Algorithms↗
PubMed1993

Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus.

The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40mapk and p41mapk, from the cytosol into the nucleus. In addition, we report that p41mapk (but not p40mapk) is localized at the cell surface ruffling membrane in serum-treated cells. To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinase-negative forms of p40mapk and p41mapk. The kinase-negative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus. Together, these data demonstrate that the pattern of serum-induced redistribution of p40mapk is different from p41mapk. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

Amino Acid Sequence↗
PubMed1993

Chemotaxis of newt eosinophils: calcium regulation of chemotactic response.

Local chemical events underlying chemotaxis were characterized in a new model cell, the newt eosinophil. These cells exhibit a chemotactic response to a trypsin-sensitive component of newt serum. Ca2+ plays a role in this process, since treatments expected to diminish Ca2+ availability from the medium [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, Co2+, and verapamil], to break down transmembrane Ca2+ gradients (ionomycin), or to interfere with the function of intracellular Ca2+ stores (caffeine and neomycin) inhibited cell polarization and movement. Using imaging techniques we found that cytosolic Ca2+ concentration ([Ca2+]i) increased in response to newt serum. Migrating newt eosinophils exhibited a dynamic heterogeneous distribution of [Ca2+]i. [Ca2+]i was elevated in cells undergoing a change of direction relative to cells migrating persistently in one direction. Migrating cells contained gradients of [Ca2+]i along their long axis, with the front of the cell having consistently lower [Ca2+]i than the rear. When cells were loaded with the cell-permeant form of fura 2, fura 2 acetoxymethyl ester, a caffeine-sensitive membrane-delimited region of elevated [Ca2+]i was seen associated with the microtubule organizing center. A model is proposed relating the distribution of [Ca2+]i and the location of the external stimulus to the generation and interaction of substances within the cell that both simulate and inhibit increases in [Ca2+]i.

Amino Acid Sequence↗
PubMed1992

Primary structure required for the inhibition of smooth muscle myosin light chain kinase.

Myosin light chain kinase (MLCK) contains the autoinhibitor sequence right next to the N-terminus side of the calmodulin binding region. In this paper, the structural requirement of the inhibition of MLCK activity was studied using synthetic peptide analogs. Peptides Ala-783-Lys-799 and Ala-783-Arg-798 inhibited calmodulin independent MLCK at the same potency as the peptide Ala-783-Gly-804. Deletion of Arg-797-Lys-799 or substitution of these residues to Ala markedly increased the Ki while the substitution of Lys-792 and Lys-793 to Ala and the deletion of Lys-784-Lys-785 did not affect the inhibitory activity of the peptides. The results suggest that Arg-797-Arg-798 are especially important for the inhibitory activity among other basic residues in the autoinhibitory region.

Amino Acid Sequence↗
PubMed1992

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.

Amino Acid Sequence↗
PubMed1992

A yeast splicing factor is localized in discrete subnuclear domains.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.

Blotting, Western↗
PubMed1992

Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy.

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of non-poly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).

Actin Cytoskeleton↗
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

Calcium homeostasis in single intact smooth muscle cells.

We have demonstrated that ISO produces part of its negative inotropic action through activation of the plasmalemmal Na+/K+ pump, and reduction of [Na+]i. This action is mediated by the beta-adrenergic receptor through activation of adenylate cyclase. The reduction of [Na+]i is most probably translated to a change in the contractile state of the cell through activation of the Na+/Ca2+ exchanger. While the exchanger is at equilibrium when the cell is at rest, after ISO it would extrude Ca2+ at the expense of the increased Na+ gradient, resulting in a decrease Ca2+ availability and a reduction in the magnitude of subsequent contractions. We have also seen that the previous calcium history of the myoplasm can influence the time course of future calcium transients. Prolonged large increases in [Ca2+]i can accelerate the rate of its removal and depress basal [Ca2+]i levels. This action is most probably mediated through a Ca2+/calmodulin dependent protein kinase. We have observed that MLCK is both necessary and sufficient to produce contraction of Bufo marinus stomach smooth muscle. There is also evidence that an as yet unidentified Ca(2+)-calmodulin dependent protein kinase is acting to limit the magnitude and the duration of the Ca2+ transient by feeding back on processes involved in Ca2+ signal generation.

Animals↗
PubMed1991

Ca2+ movement in smooth muscle cells studied with one- and two-dimensional diffusion models.

Although many of the processes involved in the regulation of Ca2+ in smooth muscle have been studied separately, it is still not well known how they are integrated into an overall regulatory system. To examine this question and to study the time course and spatial distribution of Ca2+ in cells after activation, one- and two-dimensional diffusion models of the cell that included the major processes thought to be involved in Ca regulation were developed. The models included terms describing Ca influx, buffering, plasma membrane extrusion, and release and reuptake by the sarcoplasmic reticulum. When possible these processes were described with known parameters. Simulations with the models indicated that the sarcoplasmic reticulum Ca pump is probably primarily responsible for the removal of cytoplasmic Ca2+ after cell activation. The plasma membrane Ca-ATPase and Na/Ca exchange appeared more likely to be involved in the long term regulation of Ca2+. Pumping processes in general had little influence on the rate of rise of Ca transients. The models also showed that spatial inhomogeneities in Ca2+ probably occur in cells during the spread of the Ca signal following activation and during the subsequent return of Ca2+ to its resting level.

Animals↗
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↗
PubMed1991

Effects of putative modulators of relaxation microinjected into intact amphibian smooth muscle cells.

1. Single smooth muscle cells were isolated intact from the stomach of the toad Bufo marinus. The relaxation of cells following cessation of electrical stimulation was compared with those relaxed by pressure microinjection of either metal ion chelators or cyclic nucleotides. 2. Injection of either a Ca2+ chelator or 3',5'-cyclic AMP slowed or halted shortening and promoted re-extension of a cell or collapse of membrane evaginations (blebs) in a manner similar to that following cessation of electrical stimulation. Collapse of blebs occurred first and was then followed smoothly by the next stage with cells re-extending at maximum rates in one of three ranges at 22 degrees C. These rates, in order of increasing speed, were 0.005, 0.009 and 0.03 cell lengths s-1 after electrical stimulation, 3',5'-cyclic AMP and EDTA injection, respectively. On the other hand, shortening began at a maximum rate of about 0.1 cell lengths s-1 unless a Ca2+ chelator or 3',5'-cyclic AMP was injected about 30 s or less before electrical stimulation. Injection of these agents reduced the speed of shortening by about half. 3. Injection of a liquid per se (e.g. 140 mM-KCl) neither altered action potentials nor duplicated the changes produced by the aforementioned relaxing agents. Large, sustained injections of substances that were not relaxing agents (e.g. dilute KCl) ruptured the membrane without producing any bleb collapse or re-extension of a contracted cell. Blebs not only collapsed rapidly when a relaxing agent was injected but bleb collapse was a much more sensitive indication of relaxation than cell re-extension; small injections of relaxing agents could clearly collapse blebs with no associated measurable change in cell length. This supports the idea previously inferred from fixed or permeabilized cells, that filaments in smooth muscle are organized to produce force over short distances at points along the cell membrane, in addition to shortening along the long axis. 4. Physiological relaxation of smooth muscle can evidently be mimicked by 3',5'-cyclic AMP elevation. Restoring forces may develop during shortening of isolated smooth muscle cells in elements of their cytoskeleton, surface membrane, or contractile filaments. However, these putative forces may not be able to produce physiological re-extension in the absence of a rise in cyclic AMP and/or a fall in [Ca2+].

Animals↗
PubMed1991

Adenosine reduces the Ca2+ transients of isoproterenol-stimulated rat ventricular myocytes.

Adenosine in the heart attenuates the contractile and metabolic effects of beta-adrenergic stimulation. The effect of adenosine on changes in intracellular Ca2+ concentration [( Ca2+]i) elicited with electrical stimulation was studied in rat ventricular myocytes in the absence and presence of isoproterenol (ISO). Fura-2 was utilized as a Ca2+ indicator. Autofluorescence was determined, and in vivo calibration was conducted, for each myocyte. Phenylisopropyladenosine (PIA; 10(-7) M; 5 min), an adenosine A1 receptor agonist, had no effect on the Ca2+ transient magnitude (TM) or the rate of Ca2+ transient decline determined at 150 nM Ca2+(i) (RD150). ISO (10(-8) M; 1 min) in the continued presence of PIA resulted in a 16% increase in the TM, but no change in the RD150. Inhibiting the PIA with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10(-7) M; 3 min) in the continued presence of ISO plus PIA resulted in a further 51% increase in the TM and a 57% increase in the RD150. In PIA-treated myocytes, ISO-induced spontaneous high-frequency Ca2+ transients occasionally were observed after the inhibition of PIA by DPCPX. The results of this study suggest that adenosine attenuates myocardial contractile responses to beta-adrenergic stimulation, in part, by reducing the beta-adrenergic-induced changes in the Ca2+ transients occurring in the contracting ventricular myocyte.

Adenosine↗
PubMed1990

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium.

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction.

To examine the importance of myosin light chain kinase (MLCK) in the initiation of contraction in smooth muscle, we used a constitutively active form of MLCK (IMLCK) and two specific peptide inhibitors of MLCK to study the activation of skinned single smooth muscle cells. Although unregulated by Ca-calmodulin, IMLCK, in vitro, was found to have biochemical properties like those of MLCK. Upon photolysis of caged ATP, IMLCK caused Ca-free shortening of skinned cells similar in time course and extent to that induced by Ca2+. Two peptide probes, RS-20 and SM-1, patterned after the Ca-calmodulin binding site and a pseudosubstrate inhibitory site, respectively, of the native MLCK molecule, were shown to specifically inhibit MLCK in in vitro experiments. Both peptides dose dependently inhibited Ca-induced shortening of skinned single cells. These results indicate that MLCK plays an essential role in the activation process in the smooth muscle cell in that activation of this enzyme is both necessary and sufficient for the initiation of contraction.

Actomyosin↗