Interplay between desolvation and secondary structure in mediating cosolvent and temperature induced alpha-synuclein aggregation.

Both increased temperature and moderate concentrations of fluorinated alcohols enhance aggregation of the Parkinson's disease-associated protein α-synuclein (αS). Here, we investigate the secondary structural rearrangements induced by heating and trifluoroethanol [TFE]. At low TFE concentrations, CD spectra feature a negative peak characteristic of disordered polypeptides near 200 nm and a slight shoulder around 220 nm suggesting some polyproline-II content. Upon heating, these peaks weaken, while a weak negative signal develops at 222 nm. At high TFE concentrations, the spectra show distinct minima at 208 and 222 nm, indicative of considerable α-helical structure, which diminish upon heating. We observe a crossover between the low-TFE and high-TFE behavior near 15% TFE, where we previously showed that a partially helical intermediate is populated. We postulate that the protein is well solvated by water at low TFE concentrations and by TFE at high TFE concentrations, but may become desolvated at the crossover point. We discuss the potential roles and interplay of desolvation and helical secondary structure in driving αS aggregation.

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.

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.

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.

Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells.

We have recently shown that small oligomers of IgE bound to univalent receptors for IgE on the surface of rat basophilic leukemia cells induce extensive aggregation of the receptors at 4 degrees C into patches resolvable by fluorescence microscopy and that this does not occur with monomeric IgE (Menon, A. K., D. Holowka, and B. Baird, 1984, J. Cell Biol. 98:577-583). Here we use fluorescence photobleaching recovery measurements to show that receptor oligomerization by this means is accompanied by a dramatic reduction of receptor lateral mobility, and that this immobilization occurs even when the clustering is not microscopically detectable. Furthermore, the degree of immobility induced by a particular oligomer fraction from a gel filtration column correlates positively with its ability to trigger cellular degranulation, whereas receptors labeled with monomeric IgE have no triggering activity and exhibit typical membrane protein mobility. The slow, large-scale oligomer-induced clustering appears to be a long term consequence of earlier selective interactions that result in receptor immobilization, and this highly clustered state provides a competent, noninhibitory triggering signal resulting in cellular degranulation upon warming to 37 degrees C. We conclude that even limited clustering of IgE receptors on rat basophilic leukemia cells induces interactions with other cellular components that constrain receptor mobility and eventually cause massive coalescence of the clusters. These primary selective interactions occurring at the level of receptor oligomers or small clusters of oligomers that result in immobilization may play a role in triggering cellular degranulation.

Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization.

Controlled cross-linking of IgE-receptor complexes on the surface of rat basophilic leukemia cells and mast cells has allowed a comparison of the lateral mobility and cell triggering activity of monomers, dimers, and higher oligomers of receptors. Addition of a monoclonal anti-IgE(Fc) antibody to IgE-sensitized cells in stoichiometric amounts relative to IgE produces IgE-receptor dimers with high efficiency. These dimers are nearly as mobile as IgE-receptor monomers and trigger cellular degranulation poorly, but in the presence of 30% D2O, substantial immobilization of the dimers is seen and degranulation activity doubles. Addition of this monoclonal antibody in larger amounts results in the formation of larger oligomeric receptor clusters which are immobile and effectively trigger the cells. Thus, small receptor clusters that are active in stimulating degranulation are immobilized in a process that is not anticipated by simple hydrodynamic theories. Further experiments involving cross-linking of receptor-bound IgE by multivalent antigen demonstrate that immobilization of receptors occurs rapidly (less than 2 min) upon cross-linking and is fully and rapidly reversible by the addition of excess monovalent hapten. The rapidity and reversibility of the immobilization process are entirely consistent with the possibility that immobilization represents a recognition event between clustered receptors and cytoskeleton-associated components that plays an important role early in the cell triggering mechanism.

Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

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.

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.

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.

Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.

Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.

Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts.

The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.

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.

Two-photon laser scanning fluorescence microscopy.

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

Molecular crowding on the cell surface.

Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions of proteins in applied potential gradients. The fraction of accessible surface occupied by mobile surface proteins can be accurately represented by including steric exclusion in the statistical thermodynamic analysis of the data. The analyses revealed enhanced, concentration-dependent activity coefficients, implying unanticipated thermodynamic activity even at typical cell surface receptor concentrations.

Measuring serotonin distribution in live cells with three-photon excitation.

Tryptophan and serotonin were imaged with infrared illumination by three-photon excitation (3PE) of their native ultraviolet (UV) fluorescence. This technique, established by 3PE cross section measurements of tryptophan and the monoamines serotonin and dopamine, circumvents the limitations imposed by photodamage, scattering, and indiscriminate background encountered in other UV microscopies. Three-dimensionally resolved images are presented along with measurements of the serotonin concentration ( approximately 50 mM) and content (up to approximately 5 x 10(8) molecules) of individual secretory granules.

Exchange of protein molecules through connections between higher plant plastids.

Individual plastids of vascular plants have generally been considered to be discrete autonomous entities that do not directly communicate with each other. However, in transgenic plants in which the plastid stroma was labeled with green fluorescent protein (GFP), thin tubular projections emanated from individual plastids and sometimes connected to other plastids. Flow of GFP between interconnected plastids could be observed when a single plastid or an interconnecting plastid tubule was photobleached and the loss of green fluorescence by both plastids was seen. These tubules allow the exchange of molecules within an interplastid communication system, which may facilitate the coordination of plastid activities.

Lateral transport of a lipid probe and labeled proteins on a cell membrane.

Diffusion coefficients (D) of a lipid probe and labeled proteins on L-6 myoblast membranes have been measured giving D(protein) approximately 2 X 10(-10) square centimeter per second and D (lipid probe) approximately 9 X 10(-9) square centimeter per second. Some of the membrane proteins are immobile, but the lipid probe diffuses freely over macroscopic distances. Cytochalasin B slows protein but not lipid probe diffusion.

Auditory membrane vibrations: measurements at sub-angstrom levels by optical heterodyne spectroscopy.

We describe an optical technique for measurement of mechanical vibrations in the auditory organs of living animals. The technique uses light scattered from the vibrating structure and offers several new advantages. Better than 1 angstrom sensitivity, 10 micrometers spatial resolution, and > 70 decibels dynamic range are achieved. Illustrative measurements of the mechanical response of the tympanic membrane of crickets (Gryllidae) are reported.

Lateral diffusion in planar lipid bilayers.

Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.