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D L Taylor

Publications and source records attributed to D L Taylor.

At least 145 records · Page 8Linked to original sources
PubMed1987

Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells.

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.

Animals↗
PubMed1987

Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

Animals↗
PubMed1987

Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells.

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.

Actins↗
PubMed1986

New cyanomorpholinyl byproduct of doxorubicin reductive alkylation.

Previously we reported that reductive alkylation of doxorubicin with 2,2'-oxybis[acetaldehyde] and NaBH3CN to form the 4''-morpholinyl derivative also gave the intensely potent 3''-cyano-4''-morpholinyl as a byproduct, by addition of CN- to an iminium intermediate in place of hydride. We now find that sugar 4'-OH is a third nucleophile that can add to the iminium intermediate in this reaction. Bridging of the 4'-OH to the morpholine ring at C.5'' formed a novel byproduct with an oxazolidino ring fused to the sugar and morpholine. The new product was minor at neutral pH but predominant at an acidic pH. When tested against tumors in mice it was 4-6 times more potent than doxorubicin. Hence, in comparison with the 3''-cyano-4''-morpholinyl, potency was reduced up to 100-fold by the O bridge. Analytical HPLC showed the presence of three of the four possible diastereoisomers, and two were isolated. The diastereoisomers appeared to differ in stability. In vitro tests suggested that biological potency varied inversely with stability.

Alkylation↗
PubMed1986

N-(cyanomethyl)- and N-(2-methoxy-1-cyanoethyl)anthracyclines and related carboxyl derivatives.

Treatment of doxorubicin with formaldehyde and NaCN afforded the N-(cyanomethyl) derivative as a stable alpha-cyanoamine with but moderate antitumor activity in mice, although it was prototypal to the intensely potent alpha-cyanomorpholine derivative. 2-Methoxyacetaldehyde and NaCN afforded the N-(2-methoxy-1-cyanoethyl) derivative as an open-chain analogue of the cyanomorpholine. This analogue underwent rapid hydrolysis to doxorubicin and appeared to act as a prodrug, giving increased antitumor efficacy although with decreased potency. N-(Carboxymethyl)daunorubicin was a highly water-soluble but inactive analogue, synthesized by N-alkylation with ethyl iodoacetate and saponification. The similar N-alkylation of N-(cyanomethyl) daunorubicin demonstrated the combining of N-alkyl chains having different functional substituents.

Animals↗
PubMed1986

N-(2-hydroxyethyl)doxorubicin from hydrolysis of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin.

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

Animals↗
PubMed1986

Probing the mechanism of incorporation of fluorescently labeled actin into stress fibers.

The mechanism of actin incorporation into and association with stress fibers of 3T3 and WI38 fibroblasts was examined by fluorescent analog cytochemistry, fluorescence recovery after photobleaching (FRAP), image analysis, and immunoelectron microscopy. Microinjected, fluorescein-labeled actin (AF-actin) became associated with stress fibers as early as 5 min post-injection. There was no detectable cellular polarity in the association of AF-actin with pre-existing stress fibers relative to perinuclear or peripheral regions. The rate of incorporation was quantified by image analysis of images generated with a two-dimensional photon counting microchannel plate camera. After equilibration of up to 2 h post-injection, FRAP demonstrated that actin subunits exchanged rapidly between filaments in stress fibers and the surrounding cytoplasm. When co-injected with rhodamine-labeled bovine serum albumin as a control, only actin was detected in the phase-dense stress fibers. The control protein was excluded from fibers and any linear fluorescence of the control was demonstrated as a pathlength artifact. The incorporation of AF-actin into stress fibers was studied by immunoelectron microscopy using anti-fluorescein as the primary antibody and goat anti-rabbit IgG coupled to peroxidase as the secondary antibody. At 5 min post-injection, reaction product was localized periodically in some fibers with a periodicity of approximately 0.75 microns. In large diameter fibers at 5 min post-injection, the analog was seen first on the surface of fibers, with individual filaments resolvable within the core. In the same cell, thinner diameter fibers were labeled uniformly throughout the diameter. By 20 min post-injection, most fibers were uniformly labeled. We conclude that the rate of actin subunit exchange in vivo is extremely rapid with molecular incorporation into actin filaments of stress fibers occurring as early as a few minutes post-injection. Exchange appears to first occur in filaments along the surface of stress fibers and then into more central regions in a periodic manner. We suggest that the periodic localization of actin at very early time points is due to a local microheterogeneity in which microdomains of fast vs. slower incorporation result from the periodic localization of actin-binding protein, such as alpha-actinin, along the length of the fiber.

Actin Cytoskeleton↗
PubMed1986

Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i.

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In contrast, a rise in [Ca++]i of macrophages was measured during phagocytosis occurring in a serum-free saline of pH 7.85, and as a consequence of incubation with quin2 A/M. We estimate that had a change in [Ca++]i occurred during phagocytosis, aequorin would have detected a rise from 0.1 to 1.0 microM taking place in as little as 2% of the macrophage's cytoplasmic volume. We therefore suggest that either Ca++ is not involved as a cytoplasmic signal for phagocytosis or that increases in [Ca++]i during phagocytosis are confined to such small regions of cytoplasm as to be below the limits of detection by our cellular averaging method. Our data emphasizes, moreover, the need for well-defined, nonperturbing conditions in such measurements of [Ca++]i.

Aequorin↗
PubMed1986

Probing the structure of cytoplasm.

We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.

Animals↗
PubMed1986

Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells.

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.

Cytoplasm↗
PubMed1985

Light-scattering changes during chemotactic stimulation of human neutrophils: kinetics followed by flow cytometry.

The light-scattering properties of human neutrophils were compared on a cell-by-cell basis before and after stimulation with chemotactic peptide using flow cytometry. Between 20 and 180 sec after peptide addition, side (90 degrees) scatter declined by up to 4% and forward scatter increased up to 6%. Between 3 and 15 min, side scatter increased up to 15% and forward scatter decreased up to 5%. Association of a fluorescence chemoattractant with neutrophils was most rapid during the initial phase of increasing forward and decreasing side scatter, and association saturated before the maximum increase in side scatter. Evidence is presented that the observed changes in scatter were not a consequence of chemoattractant-induced cell-cell adhesion or neutrophil degranulation. Rather, the early phases of light-scattering changes are interpreted to represent membrane ruffling by the stimulated neutrophil; the later phases polarization of the neutrophil morphology.

Chemotaxis, Leukocyte↗
PubMed1985

Comparative cytotoxicities of various morpholinyl anthracyclines.

A series of quinone- and sugar-modified analogs of adriamycin have been tested for growth inhibition of adriamycin-sensitive (P388/S) and -resistant (P388/ADR) sublines of P388 murine leukemia cells in vitro. P388/ADR is less resistant to analogs of adriamycin containing either a 3'-deamino-3'-(4"-morpholinyl) group, MRA; or a -(3"-cyano-4"-morpholinyl) group, MRA-CN, than to adriamycin. However, MRA-CN was the most potent growth inhibitor of either subline. This potency is reduced by either modification of the quinone unit with a 5-imino substituent or restriction of the cyano-morpholinyl ring by an oxygen bridge to the daunosamine sugar. The calcium antagonist verapamil substantially increases the cytotoxicity of adriamycin to P388/ADR but has no appreciable effect on the cytotoxicity of either MRA or MRA-CN. The results suggest that increased uptake and retention by both MRA and MRA-CN may contribute to their increased cytotoxicity, but that the intense potency of the cyano-morpholinyl analogs must be due to other unique properties of these compounds.

Animals↗
PubMed1985

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

Actins↗
PubMed1985

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate.

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.

Aequorin↗
PubMed1985

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells.

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.

Actins↗