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  • 1.
    Bergstrand, Jan
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Wennmalm, Stefan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Scanning inverse fluorescence correlation spectroscopy2014In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 22, no 11, p. 13073-13090Article in journal (Refereed)
    Abstract [en]

    Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

  • 2.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ronnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Bondar, Alexander
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy2011In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 12, p. 16-Article in journal (Refereed)
    Abstract [en]

    Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (alpha 3 isoform) in the postsynaptic region of the spine. Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

  • 3.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, L.
    Westin, L.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aperia, A.
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatial Distribution of DARPP-32 in Dendritic Spines2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e75155-Article in journal (Refereed)
    Abstract [en]

    The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3́, 5́-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons. Primary culture of striatal neurons were immunofluorescently labeled for DARPP-32 with Alexa-594 and for the dopamine D1 receptor (D1R) with atto-647N. Dual-color STED microscopy revealed discrete localizations of DARPP-32 and D1R in the spine structure, with clustered distributions in both head and neck. Dissected spine structures reveal that the DARPP-32 signal rarely overlapped with the D1R signal. The D1R receptor is positioned in an "aggregated" manner primarily in the spine head and to some extent in the neck, while DARPP-32 forms several neighboring small nanoclusters spanning the whole spine structure. The DARPP-32 clusters have a mean size of 52 +/- 6 nm, which is close to the resolution limit of the microscope and corresponds to the physical size of a few individual phosphoprotein immunocomplexes. Dissection of synaptic proteins using superresolution microscopy gives possibilities to reveal in better detail biologically relevant information, as compared to diffraction-limited microscopy. In this work, the dissected postsynaptic topology of the DARPP-32 phosphoprotein provides strong evidence for a compartmentalized and confined distribution in dendritic spines. The protein topology and the relatively low copy number of phosphoprotein provides a conception of DARPP-32's possibilities to fine-tune the regulation of synaptic signaling, which should have an impact on the performance of the neuronal circuits in which it is expressed.

  • 4.
    Blom, Hans
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Scott, Lena
    Spicarova, Zuzana
    Rantanen, Ville
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aperia, Anita
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Nearest neighbor analysis of dopamine D1 receptors and Na plus -K plus -ATPases in dendritic spines dissected by STED microscopy2012In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 75, no 2, p. 220-228Article in journal (Refereed)
    Abstract [en]

    Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na+,K+-ATPase. The analysis gave new information on how dense the D1 receptor and Na+,K+-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na+,K+-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes. Microsc. Res. Tech., 2011.

  • 5. Chen, Xingqi
    et al.
    Shi, Chengxi
    Yammine, Samer
    Göndör, Anita
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fernandez-Woodbridge, Alejandro
    Sumida, Noriyuki
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Ohlsson, Rolf
    Chromatin in situ proximity (ChrISP): Single-cell analysis of chromatin proximities at a high resolution2014In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 56, no 3, p. 117-124Article in journal (Refereed)
    Abstract [en]

    Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of similar to 170 angstrom in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA.with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

  • 6. Gad, Annica K. B.
    et al.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spaar, Alexander
    Savchenko, Andrii A.
    Petranyi, Gabor
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Szekely, Laszlo
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 6, p. 2374-2382Article in journal (Refereed)
    Abstract [en]

    The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.-Gad, A. K. B., Ronnlund, D., Spaar, A., Savchenko, A. A., Petranyi, G., Blom, H., Szekely, L., Widengren, J., Aspenstrom, P. Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.

  • 7. Larhammar, Martin
    et al.
    Patra, Kalicharan
    Blunder, Martina
    Emilsson, Lina
    Peuckert, Christiane
    Arvidsson, Emma
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Preobraschenski, Julia
    Birgner, Carolina
    Limbach, Christoph
    Widengren, Jerker
    Blom, Hans
    Jahn, Reinhard
    Wallen-Mackenzie, Asa
    Kullander, Klas
    SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis2015In: Biological Psychiatry, ISSN 0006-3223, E-ISSN 1873-2402, Vol. 77, no 6, p. 526-536Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis. METHODS: We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine- regulated behaviors. RESULTS: We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice over-expressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine. CONCLUSIONS: Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

  • 8. Mellroth, Peter
    et al.
    Daniels, Robert
    Eberhardt, Alice
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Normark, Staffan
    Henriques-Normark, Birgitta
    LytA, Major Autolysin of Streptococcus pneumoniae, Requires Access to Nascent Peptidoglycan2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 14, p. 11018-11029Article in journal (Refereed)
    Abstract [en]

    Background: The regulation of cell wall hydrolysis by the pneumococcal autolysin LytA is poorly understood. Results: The cell wall is susceptible to extracellular LytA only during the stationary phase or after cell wall synthesis inhibition. Conclusion: LytA is regulated on the substrate level, where peptidoglycan modifications likely prevent LytA hydrolysis. Significance: The control of amidases is essential for bacterial survival, cell-wall synthesis, and division.

  • 9. Mocsar, Gabor
    et al.
    Volko, Julianna
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Nagy, Peter
    Szollosi, Janos
    Toth, Katalin
    Goldman, Carolyn K.
    Damjanovich, Sandor
    Waldmann, Thomas A.
    Bodnar, Andrea
    Vamosi, Gyorgy
    MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and-15 Receptors in T Cells2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 1, p. 100-112Article in journal (Refereed)
    Abstract [en]

    MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Forster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2R alpha/IL-15R alpha. The mobility of not only MHC I but also that of IL-2R alpha/IL-15R alpha increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400-600 to 200-300 nm, whereas those of IL-2R alpha/IL-15R alpha hardly changed. MHC I and IL-2R alpha/IL-15R alpha colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1-and IL-2R alpha/IL-15R alpha-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins.

  • 10.
    Nordgren, Niklas
    et al.
    KTH, School of Chemical Science and Engineering (CHE).
    Rathje, Li-Sophie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE).
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Chemical Science and Engineering (CHE).
    Aspenstrom, Pontus
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Gad, Annica
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Oncogene induced stiffening of living cells2015In: Abstracts of Papers of the American Chemical Society, ISSN 0065-7727, Vol. 249Article in journal (Other academic)
  • 11. Rathje, Li-Sophie Z.
    et al.
    Nordgren, Niklas
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Coating Technology.
    Pettersson, Torbjörn
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Fibre Technology.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Gad, Annica K. B.
    Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 4, p. 1515-1520Article in journal (Refereed)
    Abstract [en]

    Oncogenes deregulate fundamental cellular functions, which can lead to development of tumors, tumor-cell invasion, and metastasis. As the mechanical properties of cells govern cell motility, we hypothesized that oncogenes promote cell invasion by inducing cytoskeletal changes that increase cellular stiffness. We show that the oncogenes simian virus 40 large T antigen, c-Myc, and cyclin E induce spatial reorganization of the vimentin intermediate filament network in cells. At the cellular level, this reorganization manifests as increased width of vimentin fibers and the collapse of the vimentin network. At nanoscale resolution, the organization of vimentin fibers in these oncogene-expressing cells was more entangled, with increased width of the fibers compared with control cells. Expression of these oncogenes also resulted in up-regulation of the tubulin deacetylase histone deacetylase 6 (HDAC6) and altered spatial distribution of acetylated microtubules. This oncogene expression also induced increases in cellular stiffness and promoted the invasive capacity of the cells. Importantly, HDAC6 was required and sufficient for the structural collapse of the vimentin filament network, and was required for increased cellular stiffness of the oncogene-expressing cells. Taken together, these data are consistent with the possibility that oncogenes can induce cellular stiffness via an HDAC6-induced reorganization of the vimentin intermediate filament network.

  • 12.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Super resolution optical imaging – image analysis, multicolor development and biological applications2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis focuses on super resolution STED optical imaging. STED provides a wealth of new informational content to the acquired images by using stimulated emission to surpass the diffraction limit in optical fluorescence microscopy. To further increase the informational content, a new method to perform multicolor STED imaging by exploiting differences in the photostability and excitation spectra of dyes is presented. In order to extract information from the images, computational algorithms which handle the new type of high resolution informational content are developed.

    We propose that multicolor super resolution imaging in combination with image analysis can reduce the amount of clinical samples required to perform accurate cancer diagnosis. To date, such diagnosis is based mainly on significant amounts of tissue samples extracted from the suspected tumor site. The sample extraction often requires anesthetics and can lead to complications such as hematoma, infections and even cancer cell ceding along the needle track. We show that by applying multicolor STED and image analysis, the information gained from single cells is greatly increased. We therefore propose that accurate diagnosis can be based on significantly less extracted tissue material, allowing for a more patient friendly sampling. This approach can also be applied when studying blood platelets, where we show how the high informational content can be used to identify platelet specific activational states. Since platelets are involved in many different types of diseases, such analysis could provide means of performing truly minimally invasive diagnostics based on a simple blood test.

    In addition, our data makes it possible to understand in finer detail the underlying mechanisms rendering cells metastasis competent. We combine the high resolution spatial information provided by STED with information regarding the adhesive forces of cells measured by TFM (Traction Force Microscopy) and the cell stiffness measured by AFM (Atomic Force Microscopy). Such comparisons provide a link between the specific highly resolved protein distributions and different cellular mechanics and functions.

    This thesis also includes STED imaging and analysis on the spatial organization of neuronal synaptic regulating proteins, implicating the speed with which neuronal signaling can be regulated.

  • 13.
    Rönnlund, Daniel
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Gad, Annica K. B.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Spatial organization of proteins in metastasizing cells2013In: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 83, no 9, p. 855-865Article in journal (Refereed)
    Abstract [en]

    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.

  • 14.
    Rönnlund, Daniel
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Xu, Lei
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Perols, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Eriksson Karlström, Amelie
    KTH, School of Biotechnology (BIO), Protein Technology.
    Auer, Gert
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Multicolor Fluorescence Nanoscopy by Photobleaching: Concept Verification and its Application to Resolve Selective Storage of Proteins in Platelets2014In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 8, no 5, p. 4358-4365Article in journal (Refereed)
    Abstract [en]

    Fluorescence nanoscopy provides means to discernthe finer details of protein localization and interaction in cells by offeringan order of magnitude higher resolution than conventional optical imagingtechniques. However, these super resolution techniques put higher demands onthe optical system as well as on the fluorescent probes, making multicolorfluorescence nanoscopy a challenging task. Here we present a new and simpleprocedure which exploits the photostability and excitation spectra of dyes toincrease the number of simultaneous recordable targets in STED nanoscopy. Weuse this procedure to demonstrate four color STED imaging of platelets with ≤40 nm resolution and low crosstalk. Platelets can selectively store, sequesterand release a multitude of different proteins, and in a manner specific fordifferent physiological and disease states. By applying multicolor nanoscopy tostudy platelets, we can achieve spatial mapping of the protein organizationwith a high resolution, for multiple proteins at the same time and in the samecell. This provides a means to identify specific platelet activation states fordiagnostic purposes and to understand the underlying protein storage andrelease mechanisms. We studied the organization of the pro- and anti-angiogenicproteins VEGF and PF-4 together with fibrinogen and filamentous actin, andfound distinct features in their respective protein localization. Further,colocalization analysis revealed only minor overlap between the proteins VEGFand PF-4 indicating that they have separate storage and release mechanisms,corresponding well with their opposite rules as pro- and anti-angiogenicproteins, respectively.

  • 15.
    Rönnlund, Daniel
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Yang, Yang
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Blom, Hans
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Auer, Gert
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Fluorescence Nanoscopy of Platelets Resolves Platelet-State Specific Storage, Release and Uptake of Proteins, Opening up Future Diagnostic Applications2012In: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 1, no 6, p. 707-713Article in journal (Refereed)
    Abstract [en]

    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.

  • 16.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Braun, Laura J.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Aspenstrom, Pontus
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Gad, Annica K. B.
    Univ Madeira, CQM, Campus Penteada, P-9020105 Funchal, Portugal..
    Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence2018In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 9, p. 1635-1652Article in journal (Refereed)
    Abstract [en]

    Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

  • 17.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Braun, Laura
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Aspenström, Pontus
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Gad, Annica
    Karolinska Institutet, Department of Microbiology, Tumor and Cell Biology.
    Nanoscale analysis of cell-matrix adhesionsManuscript (preprint) (Other academic)
    Abstract [en]

    Cell adhesion to the extracellular matrix is required for physiological processes, such as morphogenesis and wound healing. The cell adheres to the extracellular matrix via focal adhesions, which are considered to be cell adhesion organelles that govern cell function. However, the spatial architecture and organization of focal adhesions at a nanometer scale remain unclear. Therefore, we compared the spatial distribution of focal adhesion components within and outside of focal adhesions, using STED microscopy with resolution of 40-50nm. Our results are consistent with the concept that at the nanometer scale, adhesion proteins within but not outside of focal adhesions are composed by nanoscale protein clusters that attach to the extracellular matrix.

  • 18.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics.
    Aspenstrom, Pontus
    Braun, Laura J.
    Gad, Annica K. B.
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Resolution, target density and labeling effects in colocalization studies - suppression of false positives by nanoscopy and modified algorithms2016In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, no 5, p. 882-898Article in journal (Refereed)
    Abstract [en]

    Colocalization analyses of fluorescence images are extensively used to quantify molecular interactions in cells. In recent years, fluorescence nanoscopy has approached resolutions close to molecular dimensions. However, the extent to which image resolution influences different colocalization estimates has not been systematically investigated. In this work, we applied simulations and resolution-tunable stimulated emission depletion microscopy to evaluate how the resolution, molecular density and label size of targeted molecules influence estimates of the most commonly used colocalization algorithms (Pearson correlation coefficient, Manders' M1 and M2 coefficients), as well as estimates by the image cross-correlation spectroscopy method. We investigated the practically measureable extents of colocalization for stimulated emission depletion microscopy with positive and negative control samples with an aim to identifying the strengths and weaknesses of nanoscopic techniques for colocalization studies. At a typical optical resolution of a confocal microscope (200-300 nm), our results indicate that the extent of colocalization is typically overestimated by the tested algorithms, especially at high molecular densities. Only minor effects of this kind were observed at higher resolutions (< 60 nm). By contrast, underestimation of colocalization may occur if the resolution is close to the size of the label/affinity molecules themselves. To suppress false positives at confocal resolutions and high molecular densities, we introduce a statistical variant of Costes' threshold searching algorithm, used in combination with correlation-based methods like the Pearson coefficient and the image cross-correlation spectroscopy approach, to set intensity thresholds separating background noise from signals.

  • 19.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Gad, Annica
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Braun, Laura
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Aspenström, Pontus
    Karolinska Institute, Department of Microbiology, Tumor and Cell Biology .
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Effects of resolution, target density and labeling on co-localization estimates – nanoscopy and modified algorithms to suppress false positivesManuscript (preprint) (Other academic)
    Abstract [en]

    Co-localization analysis of fluorescence images is an important and extensively used tool to quantify molecular interactions in cells. In recent years, fluorescence nanoscopy techniques have made great progress and approach resolutions close to molecular dimensions. Although a strong increase in image resolution evidently influences different co-localization estimates it has to date not been systematically evaluated to what extent co-localization analyses can benefit from such techniques. . In this work, we apply simulations and resolution-tunable stimulated emission depletion (STED) microscopy to evaluate how resolution, molecular density and label size for the targeted molecules, as well as intensity variation and signal-to-noise ratio influence the estimates of the most commonly used co-localization algorithms (Pearson correlation coefficient, Manders M1 & M2 coefficients, and the image cross correlation spectroscopy (ICCS) method). We investigated the practically measureable extents of co-localization when using STED microscopy with positive and negative control samples and discussed the strengths and weaknesses of using nanoscopic techniques for co-localization studies. At a typical optical resolution of a confocal microscope (200-300 nm) our results indicate that the extent of co-localization is typically over-estimated by the tested algorithms, especially at high molecular densities. Only minor effects of this kind were observed at higher resolutions (< 60 nm). Apart from higher resolution, we introduced as an additional remedy a statistical variant of Costes threshold searching algorithm to set intensity thresholds separating background noise from signals. By this variant, combined with correlation-based methods like the Pearson coefficient and the ICCS approach, false positives at confocal resolutions and high molecular densities were found to be strongly suppressed.

  • 20.
    Xu, Lei
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Rönnlund, Daniel
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Gad, Annica K. B.
    Braun, Laura J.
    Aspenström, Pontus
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
    Effects of resolution, target density and labeling on co-localization estimates: nanoscopy and modified algorithms to suppress falseManuscript (preprint) (Other academic)
1 - 20 of 20
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