| 1. |
- Almqvist, Nils, et al.
(författare)
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Elasticity and adhesion force mapping reveals real-time clustering of growth factor receptors and associated changes in local cellular rheological properties
- 2004
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 86:3, s. 1753-1762
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Tidskriftsartikel (refereegranskat)abstract
- Cell surface macromolecules such as receptors and ion channels serve as the interface link between the cytoplasm and the extracellular region. Their density, distribution, and clustering are key spatial features influencing effective and proper physical and biochemical cellular responses to many regulatory signals. In this study, the effect of plasma-membrane receptor clustering on local cell mechanics was obtained from maps of interaction forces between antibody-conjugated atomic force microscope tips and a specific receptor, a vascular endothelial growth factor (VEGF) receptor. The technique allows simultaneous measurement of the real-time motion of specific macromolecules and their effect on local rheological properties like elasticity. The clustering was stimulated by online additions of VEGF, or antibody against VEGF receptors. VEGF receptors are found to concentrate toward the cell boundaries and cluster rapidly after the online additions commence. Elasticity of regions under the clusters is found to change remarkably, with order-of-magnitude stiffness reductions and fluidity increases. The local stiffness reductions are nearly proportional to. receptor density and, being concentrated near the cell edges, provide a mechanism for cell growth and angiogenesis.
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| 2. |
- Antzutkin, Oleg, et al.
(författare)
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Site-Specific Identification of Non-ß-Strand Conformations in Alzheimer's ß-Amyloid Fibrils by Solid-State NMR
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 84:5, s. 3326-3335
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Tidskriftsartikel (refereegranskat)abstract
- The most well-established structural feature of amyloid fibrils is the cross-ß motif, an extended ß-sheet structure formed by ß-strands oriented perpendicular to the long fibril axis. Direct experimental identification of non-ß-strand conformations in amyloid fibrils has not been reported previously. Here we report the results of solid-state NMR measurements on amyloid fibrils formed by the 40-residue ß-amyloid peptide associated with Alzheimer's disease (Aß1-40), prepared synthetically with pairs of 13C labels at consecutive backbone carbonyl sites. The measurements probe the peptide backbone conformation in residues 24-30, a segment where a non-ß-strand conformation has been suggested by earlier sequence analysis, cross-linking experiments, and molecular modeling. Data obtained with the fpRFDR-CT, DQCSA, and 2D MAS exchange solid-state NMR techniques, which provide independent constraints on the and backbone torsion angles between the labeled carbonyl sites, indicate non-ß-strand conformations at G25, S26, and G29. These results represent the first site-specific identification and characterization of non-ß-strand peptide conformations in an amyloid fibril
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| 3. |
- Arner, Anders, et al.
(författare)
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Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle
- 1998
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 75:4, s. 1895-1903
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Tidskriftsartikel (refereegranskat)abstract
- Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these contractions is not determined by the rate of Ca2+ removal or due to an increased Ca2+ sensitivity, but instead is limited by other processes such as deactivation by dephosphorylation or detachment of tension-bearing cross-bridges, possibly regulated by thin filament systems.
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| 4. |
- Balbach, John J., et al.
(författare)
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Supramolecular Structure in Full-Length Alzheimer's β-Amyloid Fibrils: Evidence for a Parallel β-Sheet Organization from Solid-State Nuclear Magnetic Resonance
- 2002
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 83:2, s. 1205-1216
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Tidskriftsartikel (refereegranskat)abstract
- We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1–40) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between 13C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional 13C magic-angle spinning NMR spectra of the labeled Aβ1–40 samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 ± 0.5 Å for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly β-sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of Aβ1–40, including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length β-amyloid fibrils.
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| 5. |
- Barg, Sebastian, et al.
(författare)
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Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells
- 2001
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Ingår i: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 81:6, s. 3308-3323
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Tidskriftsartikel (refereegranskat)abstract
- The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.
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| 6. |
- Chamberlain, Aaron K, et al.
(författare)
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Ultrastructural organization of amyloid fibrils by atomic force microscopy
- 2000
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 79:6, s. 3282-3293
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Tidskriftsartikel (refereegranskat)abstract
- Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.
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| 7. |
- Copello, J A, et al.
(författare)
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Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.
- 1997
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:1, s. 141-56
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Tidskriftsartikel (refereegranskat)abstract
- The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.
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| 8. |
- Edwards, Katarina, et al.
(författare)
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Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes
- 1997
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:1, s. 258-266
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.
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| 9. |
- Elinder, Fredrik, et al.
(författare)
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Role of individual surface charges of voltage-gated K channels
- 1999
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Ingår i: Biophysical Journal. - : Elsevier Science B.V., Amsterdam. - 0006-3495 .- 1542-0086. ; 77:3, s. 1358-1362
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Tidskriftsartikel (refereegranskat)abstract
- Fixed charges on the extracellular surface of voltage-gated ion channels influence the gating. In previous studies of cloned voltage-gated K channels, we found evidence that the functional surface charges are located on the peptide loop between the fifth transmembrane segment and the pore region (the S5-P loop). In the present study, we determine the role of individual charges of the S5-P loop by correlating primary structure with experimentally calculated surface potentials of the previously investigated channels. The results suggest that contributions to the surface potential at the voltage sensor of the different residues varies in an oscillating pattern, with the first residue of the N-terminal end of the S5-P loop, an absolutely conserved glutamate, contributing most. An analysis yields estimates of the distance between the residues and the voltage sensor, the first N-terminal residue being located at a distance of 5-6 Angstrom. To explain the results, a structural hypothesis, comprising an a-helical N-terminal end of the S5-P loop, is presented.
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| 10. |
- Fridberger, Anders, 1966-, et al.
(författare)
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Measuring hearing organ vibration patterns with confocal microscopy and optical flow
- 2004
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 86:1, s. 535-543
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Tidskriftsartikel (refereegranskat)abstract
- A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modi. cations to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.
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