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Publications (10 of 22) Show all publications
Bergstrand, J., Xu, L., Miao, X., Li, N., Öktem, O., Franzen, B., . . . Widengren, J. (2019). Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells. Nanoscale, 11(20), 10023-10033
Open this publication in new window or tab >>Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells
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2019 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 11, no 20, p. 10023-10033Article in journal (Refereed) Published
Abstract [en]

Protein contents in platelets are frequently changed upon tumor development and metastasis. However, how cancer cells can influence protein-selective redistribution and release within platelets, thereby promoting tumor development, remains largely elusive. With fluorescence-based super-resolution stimulated emission depletion (STED) imaging we reveal how specific proteins, implicated in tumor progression and metastasis, re-distribute within platelets, when subject to soluble activators (thrombin, adenosine diphosphate and thromboxane A2), and when incubated with cancer (MCF-7, MDA-MB-231, EFO21) or non-cancer cells (184A1, MCF10A). Upon cancer cell incubation, the cell-adhesion protein P-selectin was found to re-distribute into circular nano-structures, consistent with accumulation into the membrane of protein-storing alpha-granules within the platelets. These changes were to a significantly lesser extent, if at all, found in platelets incubated with normal cells, or in platelets subject to soluble platelet activators. From these patterns, we developed a classification procedure, whereby platelets exposed to cancer cells, to non-cancer cells, soluble activators, as well as non-activated platelets all could be identified in an automatic, objective manner. We demonstrate that STED imaging, in contrast to electron and confocal microscopy, has the necessary spatial resolution and labelling efficiency to identify protein distribution patterns in platelets and can resolve how they specifically change upon different activations. Combined with image analyses of specific protein distribution patterns within the platelets, STED imaging can thus have a role in future platelet-based cancer diagnostics and therapeutic monitoring. The presented approach can also bring further clarity into fundamental mechanisms for cancer cell-platelet interactions, and into non-contact cell-to-cell interactions in general.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-254018 (URN)10.1039/c9nr01967g (DOI)000469246100020 ()31086875 (PubMedID)
Note

Qc 20190814

Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-14Bibliographically approved
Pathak, A., Bergstrand, J., Sender, V., Spelmink, L., Aschtgen, M.-S., Muschiol, S., . . . Henriques-Normark, B. (2018). Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification. Nature Communications, 9, Article ID 3398.
Open this publication in new window or tab >>Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3398Article in journal (Refereed) Published
Abstract [en]

Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-234597 (URN)10.1038/s41467-018-05494-w (DOI)000442522100001 ()30139996 (PubMedID)2-s2.0-85052221727 (Scopus ID)
Note

QC 20180914

Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2019-04-04Bibliographically approved
Du, Z., Yu, J., Li, F., Deng, L., Wu, F., Huang, X., . . . Ren, J. (2018). In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy. Analytical Chemistry, 90(10), 6144-6151
Open this publication in new window or tab >>In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy
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2018 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed) Published
Abstract [en]

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-229014 (URN)10.1021/acs.analchem.8b00473 (DOI)000432478600026 ()29671327 (PubMedID)2-s2.0-85046367106 (Scopus ID)
Note

QC 20180531

Available from: 2018-05-31 Created: 2018-05-31 Last updated: 2019-04-04Bibliographically approved
Tornmalm, J. & Widengren, J. (2018). Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy. Methods, 140, 178-187
Open this publication in new window or tab >>Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy
2018 (English)In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 140, p. 178-187Article in journal (Refereed) Published
Abstract [en]

Transient state (TRAST) monitoring can determine population dynamics of long-lived, dark transient states of fluorescent molecules, detecting only the average fluorescence intensity from a sample, when subject to different excitation pulse trains. Like Fluorescence Correlation Spectroscopy (FCS), TRAST unites the detection sensitivity of fluorescence with the environmental sensitivity of long-lived non-fluorescent states, but does not rely on detection of stochastic fluorescence fluctuations from individual molecules. Relaxed requirements on noise suppression, detection quantum yield and time-resolution of the instrument, as well as on fluorescence brightness of the molecules studied, make TRAST broadly applicable, opening also for investigations based on less bright, auto-fluorescent molecules. In this work, we applied TRAST to study the transient state population dynamics within the auto-fluorescent coenzymes flavin adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). From the experimental TRAST data, we defined state models, and determined rate parameters for triplet state and redox transitions within FMN and FAD, stacking and un-stacking rates of external redox active quenching agents and by the adenine moiety of FAD itself. TRAST experiments were found to be well capable to resolve these transitions in FMN and FAD, and to track how the transitions are influenced by ambient oxygenation and redox conditions. This work demonstrates that TRAST provides a useful tool to follow local oxygenation and redox conditions via FMN and FAD fluorescence, and forms the basis for measurements on flavoproteins and of redox and metabolic conditions in more complex environments, such as in live cells.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2018
Keywords
Flavin, Fluorescence, Triplet state, Redox state, Oxygenation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-232422 (URN)10.1016/j.ymeth.2017.11.013 (DOI)000436917700019 ()29179988 (PubMedID)2-s2.0-85036623291 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2012-3045Swedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation, KAW 2012.0218
Note

QC 20180725

Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2019-03-11Bibliographically approved
Xu, L., Braun, L. J., Rönnlund, D., Widengren, J., Aspenstrom, P. & Gad, A. K. B. (2018). Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence. The FEBS Journal, 285(9), 1635-1652
Open this publication in new window or tab >>Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence
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2018 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, no 9, p. 1635-1652Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
co-localization, focal adhesion-related particles, focal adhesions, quantitative imaging, stimulated emission depletion microscopy
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-228269 (URN)10.1111/febs.14433 (DOI)000431678400007 ()29542240 (PubMedID)
Note

QC 20180522

Available from: 2018-05-22 Created: 2018-05-22 Last updated: 2018-05-22Bibliographically approved
Huang, B., Bergstrand, J., Duan, S., Zhan, Q., Widengren, J., Ågren, H. & Liu, H. (2018). Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals [Letter to the editor]. ACS Nano, 12, 10572-10575
Open this publication in new window or tab >>Overtone Vibrational Transition-Induced Lanthanide Excited-State Quenching in Yb3+/Er3+-Doped Upconversion Nanocrystals
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2018 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, p. 10572-10575Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Nano Technology
Identifiers
urn:nbn:se:kth:diva-246210 (URN)10.1021/acsnano.8b05095 (DOI)000451789200004 ()2-s2.0-85057253085 (Scopus ID)
Note

QC 20190318

Available from: 2019-03-16 Created: 2019-03-16 Last updated: 2019-03-20Bibliographically approved
Lomnytska, M., Pinto, R., Becker, S., Engstrom, U., Gustafsson, S., Bjorklund, C., . . . Auer, G. (2018). Platelet protein biomarker panel for ovarian cancer diagnosis. BIOMARKER RESEARCH, 6, Article ID 2.
Open this publication in new window or tab >>Platelet protein biomarker panel for ovarian cancer diagnosis
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2018 (English)In: BIOMARKER RESEARCH, ISSN 2050-7771, Vol. 6, article id 2Article in journal (Refereed) Published
Abstract [en]

Background: Platelets support cancer growth and spread making platelet proteins candidates in the search for biomarkers. Methods: Two-dimensional (2D) gel electrophoresis, Partial Least Squares Discriminant Analysis (PLS-DA), Western blot, DigiWest. Results: PLS-DA of platelet protein expression in 2D gels suggested differences between the International Federation of Gynaecology and Obstetrics (FIGO) stages III-IV of ovarian cancer, compared to benign adnexal lesions with a sensitivity of 96% and a specificity of 88%. A PLS-DA-based model correctly predicted 7 out of 8 cases of FIGO stages I-II of ovarian cancer after verification by western blot. Receiver-operator curve (ROC) analysis indicated a sensitivity of 83% and specificity of 76% at cut-off >0.5 (area under the curve (AUC) = 0.831, p < 0.0001) for detecting these cases. Validation on an independent set of samples by DigiWest with PLS-DA differentiated benign adnexal lesions and ovarian cancer, FIGO stages III-IV, with a sensitivity of 70% and a specificity of 83%. Conclusion: We identified a group of platelet protein biomarker candidates that can quantify the differential expression between ovarian cancer cases as compared to benign adnexal lesions.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Ovarian cancer, Platelet proteome, Biomarker, Liquid biopsy
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-221935 (URN)10.1186/s40364-018-0118-y (DOI)000419918200001 ()29344361 (PubMedID)
Note

QC 20180131

Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2018-01-31Bibliographically approved
Iovino, F., Engelen-Lee, J.-Y., Brouwer, M., van de Beek, D., van der Ende, A., Seron, M. V., . . . Henriques-Normark, B. (2017). pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion. Journal of Experimental Medicine, 214(6), 1619-1630
Open this publication in new window or tab >>pIgR and PEC AM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion
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2017 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 214, no 6, p. 1619-1630Article in journal (Refereed) Published
Abstract [en]

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

Place, publisher, year, edition, pages
Rockefeller University Press, 2017
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-210488 (URN)10.1084/jem.20161668 (DOI)000402863300007 ()28515075 (PubMedID)2-s2.0-85021856297 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilSwedish Foundation for Strategic Research Stockholm County Council
Note

QC 20170705

Available from: 2017-07-05 Created: 2017-07-05 Last updated: 2019-04-04Bibliographically approved
Luu, T. T., Vignesh, S., Wagner, A. K., Chrobok, M., Schlums, H., Tornmalm, J., . . . Hoglund, P. (2017). Reactive oxygen species are linked with general phosphorylation status and functional education of natural killer cells. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 295-295
Open this publication in new window or tab >>Reactive oxygen species are linked with general phosphorylation status and functional education of natural killer cells
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 295-295Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
WILEY, 2017
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-215792 (URN)000411865200113 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Note

QC 20171018

Available from: 2017-10-18 Created: 2017-10-18 Last updated: 2017-10-18Bibliographically approved
Cebula, M., Turan, I. S., Sjodin, B., Thulasingam, M., Brock, J., Chmyrov, V., . . . Morgenstern, R. (2016). Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase. Scientific Reports, 6, Article ID 38316.
Open this publication in new window or tab >>Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38316Article in journal (Refereed) Published
Abstract [en]

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-199488 (URN)10.1038/srep38316 (DOI)000389188000001 ()2-s2.0-85004000448 (Scopus ID)
Note

QC 20170119

Available from: 2017-01-19 Created: 2017-01-09 Last updated: 2017-11-29Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3200-0374

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