| 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. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Friedman, Ran, et al.
(författare)
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The role of small intraprotein cavities in the catalytic cycle of bacteriorhodopsin
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 85:2, s. 886-896
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Tidskriftsartikel (refereegranskat)abstract
- The last phase of the proton transfer cycle of bacteriorhodopsin calls for a passage of a proton from D38 to D96.This reaction utilizes a narrow shaft ;10-A˚ long that connects the two carboxylates that cross through a very hydrophobicdomain. As the shaft is too narrow to be permanently hydrated, there are two alternatives for the proton propagation into thechannel. The proton may propagate through the shaft without solvation at the expense of a high electrostatic barrier;alternatively, the shaft will expand to accommodate some water molecules, thus lowering the Born energy for the insertion ofthe charge into the protein (B. Scha¨ tzler, N. A. Dencher, J. Tittor, D. Oesterhelt, S. Yaniv-Checover, E. Nachliel, and G. Gutman,2003, Biophys. J. 84:671–686). A comparative study of nine published crystal-structures of bacteriorhodopsin identified, next tothe shaft, microcavities in the protein whose position and surrounding atoms are common to the reported structures. Some ofthe cavities either shrink or expand during the photocycle. It is argued that the plasticity of the cavities provides a working spaceneeded for the transient solvation of the shaft, thus reducing the activation energy necessary for the solvation of the shaft. Thissuggestion is corroborated by the recent observations of Klink et al. (B. U. Klink, R. Winter, M. Engelhard, and I. Chizhov, 2002,Biophys. J. 83:3490–3498) that the late phases of the photocycle (t $ 1 ms) are strongly inhibited by external pressure.
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| 8. |
- Hofsäss, Christofer, et al.
(författare)
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Molecular dynamics simulations of phospholipid bilayers with cholesterol
- 2003
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 84:4, s. 2192-206
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the microscopic interactions between cholesterol and lipids in biological membranes, we have performed a series of molecular dynamics simulations of large membranes with different levels of cholesterol content. The simulations extend to 10 ns, and were performed with hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. The bilayers contain 1024 lipids of which 0-40% were cholesterol and the rest DPPC. The effects of cholesterol on the structure and mesoscopic dynamics of the bilayer were monitored as a function of cholesterol concentration. The main effects observed are a significant ordering of the DPPC chains (as monitored by NMR type order parameters), a reduced fraction of gauche bonds, a reduced surface area per lipid, less undulations--corresponding to an increased bending modulus for the membrane, smaller area fluctuations, and a reduced lateral diffusion of DPPC-lipids as well as cholesterols.
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| 9. |
- Leitz, Guenther, et al.
(författare)
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Stress response in Caenorhabditis elegans caused by optical tweezers : wavelength, power, and time dependence
- 2002
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Ingår i: Biophysical Journal. - : Biophysical Society. - 0006-3495 .- 1542-0086. ; 82:4, s. 2224-2231
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Tidskriftsartikel (refereegranskat)abstract
- Optical tweezers have emerged as a powerful technique for micromanipulation of living cells. Although the technique often has been claimed to be nonintrusive, evidence has appeared that this is not always the case. This work presents evidence that near-infrared continuous-wave laser light from optical tweezers can produce stress in Caenorhabditis elegans. A transgenic strain of C. elegans, carrying an integrated heat-shock-responsive reporter gene, has been exposed to laser light under a variety of illumination conditions. It was found that gene expression was most often induced by light of 760 nm, and least by 810 nm. The stress response increased with laser power and irradiation time. At 810 nm, significant gene expression could be observed at 360 mW of illumination, which is more than one order of magnitude above that normally used in optical tweezers. In the 700-760-nm range, the results show that the stress response is caused by photochemical processes, whereas at 810 nm, it mainly has a photothermal origin. These results give further evidence that the 700-760-nm wavelength region is unsuitable for optical tweezers and suggest that work at 810 nm at normal laser powers does not cause stress at the cellular level.
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| 10. |
- Lindahl, E, 1972-, et al.
(författare)
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Mesoscopic undulations and thickness fluctuations in lipid bilayers from molecular dynamics simulations.
- 2000
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 79:1, s. 426-33
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Tidskriftsartikel (refereegranskat)abstract
- Molecular dynamics simulations of fully hydrated Dipalmitoylphosphatidylcholine bilayers, extending temporal and spatial scales by almost one order of magnitude, are presented. The present work reaches system sizes of 1024 lipids and times 10-60 ns. The simulations uncover significant dynamics and fluctuations on scales of several nanoseconds, and enable direct observation and spectral decomposition of both undulatory and thickness fluctuation modes. Although the former modes are strongly damped, the latter exhibit signs of oscillatory behavior. From this, it has been possible to calculate mesoscopic continuum properties in good agreement with experimental values. A bending modulus of 4 x 10(-20) J, bilayer area compressibility of 250-300 mN/m, and mode relaxation times in the nanosecond range are obtained. The theory of undulatory motions is revised and further extended to cover thickness fluctuations. Finally, it is proposed that thickness fluctuations is the explanation to the observed system-size dependence of equilibrium-projected area per lipid.
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