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W W Webb

Publications and source records attributed to W W Webb.

At least 109 records · Page 6Linked to original sources
PubMed1982

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells.

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.

Actins↗
PubMed1982

Hydrodynamic models of viscous coupling between motile myosin and endoplasm in characean algae.

Cytoplasmic streaming in characean algae is thought to be driven by interaction between stationary subcortical actin bundles and motile endoplasmic myosin. Implicit in this mechanism is a requirement for some form of coupling to transfer motive force from the moving myosin to the endoplasm. Three models of viscous coupling between myosin and endoplasm are presented here, and the hydrodynamic feasibility of each model is analyzed. The results show that individual myosinlike molecules moving along the actin bundles at reasonable velocities cannot exert enough viscous pull on the endoplasm to account for the observed streaming. Attachment of myosin to small spherical organelles improves viscous coupling to the endoplasm, but results for this model show that streaming can be generated only if the myosin-spheres move along the actin bundles in a virtual solid line at about twice the streaming velocity. In the third model, myosin is incorporated into a fibrous or membranous network or gel extending into the endoplasm. This network is pulled forward as the attached myosin slides along the actin bundles. Using network dimensions estimated from published micrographs of characean endoplasm, the results show that this system can easily generate the observed cytoplasmic streaming.

Actins↗
PubMed1982

Diffusion of low density lipoprotein-receptor complex on human fibroblasts.

Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.

Actins↗
PubMed1981

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

The distribution and polymerization state of actin in metaphase rat kangaroo cells was studied by fluorescence microscopy. Formaldehyde-fixed, acetone-extracted cells were labeled with either of two types of actin probes. The first, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin, has high affinity for F actin and does not bind monomeric G actin. The second was a conjugate of DNase I labeled with either tetramethylrhodamine or fluorescein. DNase binds with high affinity to G actin and with lesser affinity to F actin. The polymerization state of actin was deduced by comparing the fluorescence distribution of the phallacidin derivative with that of the fluorescent DNase. The results indicate that the pole-to-chromosome region of the metaphase spindle contains G actin but little if any conventional F actin. F actin is found concentrated in a diffuse distribution outside the spindle region in metaphase cells and returns to the interzone area between the chromosomes by early telophase. These results exclude spindle models for chromosomal movement that require more than about five F actin filaments per chromosome, support the hypothesis that F actin is involved in force generation for cell cleavage, and are not inconsistent with the possibility that actin outside the spindle may be involved in chromosomal movement.

Actins↗
PubMed1981

Fluorescence studies on modes of cytochalasin B and phallotoxin action on cytoplasmic streaming in Chara.

Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.

Actins↗
PubMed1981

In vivo staining of cytoskeletal actin by autointernalization of nontoxic concentrations of nitrobenzoxadiazole-phallacidin.

The blue light-excited fluorescent phallotoxin derivative nitrobenzoxadiazole phallacidin (NBD-Ph) was used to stain entire tissue culture monolayers of live L6 mouse cells and other mammalian cell lines without the aid of permeabilization treatment. Although cells tend to exclude the fluorescent toxin, reducing the internal concentration by approximately 1,000 times, some of it enters the cells, probably by pinocytosis, and stains actin structures at low intracellular NBD-Ph concentrations (approximately 5-15 nM), where cell toxicity was negligible or at least not detectable by phase-contrast microscopy. Protracted treatments with NBD-Ph did induce pharmacological responses similar to those of phalloidin. The dissociation constant for NBD-Ph with F-actin in fixed and extracted L6 cells was determined, from staining intensity measurements at various NBD-Ph concentrations, to be 1.5-2.5 x 10(-8) M.

Actins↗
PubMed1981

Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts.

The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.

Carbocyanines↗
PubMed1981

F-actin aggregates in transformed cells.

Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.

Actins↗
PubMed1981

Molecular mobility on the cell surface.

Many measurements of lateral diffusion of proteins and lipids on cell membranes and lipid model membranes have become available through application of fluorescence photobleaching recovery methods. A puzzling aspect of these results is slow diffusion and partial immobilization of protein molecules on the cell surface. Observed protein diffusion coefficients on vertebrate structural tissue cells are consistently D less than or equal to 10(-10) cm2/s, while lipid analogues diffuse with D approx. 10(-8) cm2/s. Substantial fractions of the cell membrane proteins are not diffusible. In a pure viscous membrane, theoretical fluid dynamics has suggested only small differences between lipid and protein diffusion coefficients. Measurements of protein diffusion in model membranes recently showed D less than or equal to 10(-9) cm2/s, as expected. Recent experiments on cell membranes show that uncoupling of the membrane from the cytoskeleton by formation of blebs releases the membrane protein molecules so that diffusion is enhanced to D greater than or equal to 10(-9) cm2/s and the non-diffusible fraction is eliminated.

Actins↗
PubMed1980

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes.

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.

Animals↗
PubMed1980

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts.

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.

Animals↗
PubMed1980

Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.

Actins↗
PubMed1979

Barrier filter for fluorescence microscopy of strongly autofluorescent plant tissues. Application to actin cables in Chara.

A liquid barrier filter for use in fluorescence microscopy of strongly autofluorescent plant tissues is described. The filter consists of a methanol solution of cupric chloride and ferric chloride and isolates fluorescein fluorescence from the strong red autofluorescence of photosynthetic plant tissues. Subcortical actin cables in the giant alga Chara are being visualized through use of this filter together with heavy meromyosin labeling.

Actins↗
PubMed1978

Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540.

The fluorescence and optical absorption of the membrane-staining dye merocyanine 540 (M-540) have been widely used to measure cellular transmembrane potentials. We have studied the molecular mechanisms of these optical changes by measuring the fluorescence polarization of M-540 and its response to membrane potential changes in hemispherical lipid bilayer membranes. The fluorescence responds to a potential step in two distinct time scales: a fast response with a rise time less than the instrumental capability of 6 micromilligram and a slow response with a time constant around 10(-1) s. Both response amplitudes are proportional to the amplitude of the membrane potential change and both require an asymmetrical distribution of M-540 across the membrane. The slow response is ascribed to a net change of the dye concentration in the membrane. The fast response appears to be dominated by a change in the distribution of orientations of the dye molecules in the membrane, with a concomitant perturbation of a monomer-dimer equilibrium, due to interaction of the applied electric field with the permanent molecular dipol moment of M-540. The amplitude of the fast fluorescence response is concentration dependent and can be modeled by including membrane saturation effects and the presence of a nonfluorescent dimer species in the membrane at high dye concentrations. Absorbance changes reported by other investigators are consistent with this model mechanism.

Chemical Phenomena↗
PubMed1978

Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals.

The lateral-diffusion coefficients (D) of a fluorescent lipid analogue, 3,3'-dioctadecylindotricarbocyanine (diI), have been measured in black lipid membranes (BLMs), in large (20--50-micron diameter) solvent-free bilayer and multilayer membrane vesicles, and in multilamellar liquid crystals of dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and egg lecithin. They show that D changes by several orders of magnitude at the liquid-crystal transformations of the solvent-free bilayers and multilayer. In all BLMs, D approximately 10(-7)cm2/s with only weak temperature dependence even near the putative phase-transformation temperatures, Tt. In the corresponding liquid crystals and large vesicles, D approximately 10(-8)cm2/s above Tt, decreasing by about two orders of magnitude to D approximately 10(-10)cm2/s below Tt. The changes of D in bilayer vesicles suggest that the expected liquid-crystal phase transitions from smectic A to a higher ordered state do persist in isolated bilayers. Retained solvent in black lipid membranes formed by both Mueller--Rudin and Montal--Mueller methods appears to enhance lateral diffusion. A simple method of forming small numbers of large solvent-free vesicles is described.

Crystallization↗