| 1. |
- Sjöholm, Johannes, et al.
(författare)
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The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the effect of lipid composition on interactions between cytochrome bo(3) and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d congruent to 100 nm) or giant (d congruent to 10 mu m) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be similar to 80 nm.
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| 2. |
- Sandén, Tor, et al.
(författare)
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Surface-coupled proton exchange of a membrane-bound proton acceptor
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - Washington : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:9, s. 4129-4134
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Tidskriftsartikel (refereegranskat)abstract
- Proton-transfer reactions across and at the surface of biological membranes are central for maintaining the transmembrane proton electrochemical gradients involved in cellular energy conversion. In this study, fluorescence correlation spectroscopy was used to measure the local protonation and deprotonation rates of single pH-sensitive fluorophores conjugated to liposome membranes, and the dependence of these rates on lipid composition and ion concentration. Measurements of proton exchange rates over a wide proton concentration range, using two different pH-sensitive fluorophores with different pKas, revealed two distinct proton exchange regimes. At high pH (> 8), proton association increases rapidly with increasing proton concentrations, presumably because the whole membrane acts as a proton-collecting antenna for the fluorophore. In contrast, at low pH (< 7), the increase in the proton association rate is slower and comparable to that of direct protonation of the fluorophore from the bulk solution. In the latter case, the proton exchange rates of the two fluorophores are indistinguishable, indicating that their protonation rates are determined by the local membrane environment. Measurements on membranes of different surface charge and at different ion concentrations made it possible to determine surface potentials, as well as the distance between the surface and the fluorophore. The results from this study define the conditions under which biological membranes can act as proton-collecting antennae and provide fundamental information on the relation between the membrane surface charge density and the local proton exchange kinetics.
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| 3. |
- Rönnlund, Daniel, et al.
(författare)
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Spatial organization of proteins in metastasizing cells
- 2013
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Ingår i: Cytometry Part A. - : John Wiley & Sons. - 1552-4922 .- 1552-4930. ; 83:9, s. 855-865
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Tidskriftsartikel (refereegranskat)abstract
- The ability of tumor cells to invade into the surrounding tissue is linked to defective adhesive and mechanical properties of the cells, which are regulated by cell surface adhesions and the intracellular filamentous cytoskeleton, respectively. With the aim to further reveal the underlying mechanisms and provide new strategies for early cancer diagnostics, we have used ultrahigh resolution stimulated emission depletion (STED) microscopy as a means to identify metastasizing cells, based on their subcellular protein distribution patterns reflecting their specific adhesive and mechanical properties. We have compared the spatial distribution of cell-matrix adhesion sites and the vimentin filamentous systems in a matched pair of primary, normal, and metastatic human fibroblast cells. We found that the metastatic cells showed significantly increased densities and more homogenous distributions of nanoscale adhesion-related particles. Moreover, they showed an increase in the number but reduced sizes of the areas of cell-matrix adhesion complexes. The organization of the vimentin intermediate filaments was also found to be significantly different in the metastasizing cells, showing an increased entanglement and loss of directionality. Image analysis procedures were established, allowing an objective detection and characterization of these features and distinction of metastatic cells from their normal counterparts. In conclusion, our results suggest that STED microscopy provides a novel tool to identify metastasizing cells from a very sparse number of cells, based on the altered spatial distribution of the cell-matrix adhesions and intermediate filaments.
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| 4. |
- Strömqvist, Johan, et al.
(författare)
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A modified FCCS procedure applied to Ly49A-MHC class Icis-interaction studies in cell membranes
- 2011
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 101:5, s. 1257-1269
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Tidskriftsartikel (refereegranskat)abstract
- The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.
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| 5. |
- Strömqvist, Johan, et al.
(författare)
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Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 99:11, s. 3821-3830
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Tidskriftsartikel (refereegranskat)abstract
- An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured
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| 6. |
- Chmyrov, Andriy, et al.
(författare)
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Iodide as a Fluorescence Quencher and Promoter-Mechanisms and Possible Implications
- 2010
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 114:34, s. 11282-11291
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Tidskriftsartikel (refereegranskat)abstract
- In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.
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| 7. |
- Widengren, Jerker, 1965-
(författare)
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Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging
- 2010
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Ingår i: Journal of the Royal Society Interface. - London : Royal Society Publishing. - 1742-5689 .- 1742-5662. ; 7:49, s. 1135-1144
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Forskningsöversikt (refereegranskat)abstract
- To increase read-out speed, sensitivity or specificity, an often applied strategy in fluorescence-based biomolecular spectroscopy and imaging is to simultaneously record two or more of the fluorescence parameters: intensity, lifetime, polarization or wavelength. This review highlights how additional, to-date largely unexploited, information can be extracted by monitoring long-lived, photo-induced transient states of organic dyes and their dynamics. Two major approaches are presented, where the transient state information is obtained either from fluorescence fluctuation analysis or by recording the time-averaged fluorescence response to a time-modulated excitation. The two approaches combine the detection sensitivity of the fluorescence signal with the environmental sensitivity of the long-lived transient states. For both techniques, proof-of-principle experiments are reviewed, and advantages, limitations and possible applications for biomolecular cellular biology studies are discussed.
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| 8. |
- Piguet, Joachim, 1979-
(författare)
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Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.
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| 9. |
- Rönnlund, Daniel, et al.
(författare)
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Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications
- 2012
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Ingår i: Advanced Healthcare Materials. - : Wiley-Blackwell. - 2192-2640 .- 2192-2659. ; 1:6, s. 707-713
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Tidskriftsartikel (refereegranskat)abstract
- Dysregulation of how platelets store, sequester and release specific proteins seems to be implicated in many disease states, including cancer. Dual-color immunofluorescence stimulated emission depletion (STED) microscopy with 40 nm resolution is used to map pro-angiogenic VEGF, anti-angiogenic PF-4 and fibrinogen in >300 individual platelets. This reveals that these proteins are stored in a segmented, zonal manner within regional clusters, significantly smaller than the size of an alpha-granule. No colocalization between the different proteins is observed. Upon platelet activation by thrombin or ADP, the proteins undergo significant spatial rearrangements, including alterations in the size and number of the protein clusters, and specific for a certain protein and the type of activation induced. Following these observations, a simple assignment procedure is used to show that the three distinct states of platelets (non-, ADP- and thrombin-activated) can be identified based on the average size, number and peripheral localization profiles of the regional protein clusters within the platelets. Thus, high-resolution spatial mapping of specific proteins is a useful procedure to detect and characterize deviations in the selective storage, release and uptake of these proteins in the platelets. Since these deviations seem to be specific for, and may even underlie, certain patophysiological states, these findings may have interesting diagnostic and therapeutic implications.
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| 10. |
- Spielmann, Thiemo, 1984-, et al.
(författare)
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Transient state imaging probes patterns of altered oxygen consumption in cancer cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Altered cellular metabolism plays an important role in many diseases, not least in many forms of cancer, where cellular metabolic pathways requiring lower oxygen consumption are often favored (the so-called Warburg effect). In this work, we have applied fluorescence-based transient state imaging and have exploited the environment sensitivity of long-lived dark states of fluorophores, in particular triplet state decay rates, to image the oxygen consumption of living cells. Our measurements can resolve differences in oxygen concentrations between different regions of individual cells, between different cell types, and also based on what metabolic pathways the cells use. In MCF-7 breast cancer cells, higher oxygen consumption can be detected when they rely on glutamine instead of glucose as their main metabolite, predominantly undergoing oxidative phosphorylation rather than glycolysis. By use of the high triplet yield dye Eosin Y the irradiance requirements during the measurements can be kept low. This reduces the instrumentation requirements, and harmful biological effects from high excitation doses can be avoided. Taken together, our imaging approach is widely applicable and capable of detecting subtle changes in oxygen consumption in live cells, stemming from the Warburg effect or reflecting other differences in the cellular metabolism. This may lead to new diagnostic means as well as advance our understanding of the interplay between cellular metabolism and major disease categories, such as cancer.
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