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Publications (10 of 88) Show all publications
Heldin, J., Rubin Sander, M., Leino, M., Thomsson, S., Lennartsson, J. & Söderberg, O. (2019). Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling. Experimental Cell Research, 380(1), 69-79
Open this publication in new window or tab >>Dynamin inhibitors impair platelet-derived growth factor beta-receptor dimerization and signaling
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2019 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 380, no 1, p. 69-79Article in journal (Refereed) Published
Abstract [en]

The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor beta-receptor (PDGFR-beta) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-beta preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-beta by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-beta activation, and that this link is not shared with the epidermal growth factor receptor.

Place, publisher, year, edition, pages
ELSEVIER INC, 2019
Keywords
RTK signaling, EGFR, PDGFR-beta, Dynasore, Dyngo, Dimerization
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-384986 (URN)10.1016/j.yexcr.2019.04.004 (DOI)000468124700008 ()30970237 (PubMedID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilSwedish Cancer Society, CAN 2018/425
Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-06-14Bibliographically approved
Wu, C.-C., Klaesson, A., Buskas, J., Ranefall, P., Mirzazadeh, R., Söderberg, O. & Wolf, J. B. W. (2019). In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation. Journal of Experimental Biology, 222(5)
Open this publication in new window or tab >>In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation
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2019 (English)In: Journal of Experimental Biology, ISSN 0022-0949, E-ISSN 1477-9145, Vol. 222, no 5Article in journal (Refereed) Published
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-381095 (URN)10.1242/jeb.194431 (DOI)000461414600021 ()30718374 (PubMedID)
Funder
EU, European Research Council, ERCStG-336536Swedish Research Council, 621-2013-4510Knut and Alice Wallenberg Foundation
Available from: 2019-03-08 Created: 2019-04-15 Last updated: 2019-05-07Bibliographically approved
Klaesson, A., Grannas, K., Ebai, T., Heldin, J., Koos, B., Leino, M., . . . Landegren, U. (2018). Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes. Scientific Reports, 8, Article ID 5400.
Open this publication in new window or tab >>Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5400Article in journal (Refereed) Published
Abstract [en]

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

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) Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-340077 (URN)10.1038/s41598-018-23582-1 (DOI)000428618900043 ()29599435 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568 264737 294409Swedish Foundation for Strategic Research Swedish Research Council
Note

Ola Söderberg and Ulf Landegren jointly supervised this work

Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-08-06Bibliographically approved
Löf, L., Arngården, L., Olsson-Strömberg, U., Siart, B., Jansson, M., Dahlin, J. S., . . . Kamali-Moghaddam, M. (2017). Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia. Scientific Reports, 7, 1-9, Article ID 623.
Open this publication in new window or tab >>Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed) Published
Abstract [en]

Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-319726 (URN)10.1038/s41598-017-00755-y (DOI)000398162400034 ()28377570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 294409Swedish Cancer SocietySwedish Research Council
Available from: 2017-04-07 Created: 2017-04-07 Last updated: 2017-05-15Bibliographically approved
Andersson, S., Sundberg, M., Pristovsek, N., Ibrahim, A., Jonsson, P., Katona, B., . . . Asplund, A. (2017). Insufficient antibody validation challenges oestrogen receptor beta research. Nature Communications, 8, Article ID 15840.
Open this publication in new window or tab >>Insufficient antibody validation challenges oestrogen receptor beta research
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2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15840Article in journal (Refereed) Published
Abstract [en]

The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-329670 (URN)10.1038/ncomms15840 (DOI)000403317200001 ()28643774 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2017-09-19 Created: 2017-09-19 Last updated: 2017-11-29Bibliographically approved
Beghini, A., Lazzaroni, F., Del Giacco, L., Söderberg, O., Biasci, D., Turrini, M., . . . Cairoli, R. (2016). Clinical Relevance Of Recurrent Allele-Specific Recombination Expressing The Wnt10Bivs1 Allele Variant In Acute Myeloid Leukemia. Paper presented at 21st Congress of the European-Hematology-Association, JUN 09-12, 2016, Copenhagen, DENMARK. Haematologica, 101, 668-669
Open this publication in new window or tab >>Clinical Relevance Of Recurrent Allele-Specific Recombination Expressing The Wnt10Bivs1 Allele Variant In Acute Myeloid Leukemia
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2016 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 668-669Article in journal, Meeting abstract (Other academic) Published
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-301461 (URN)000379484602430 ()
Conference
21st Congress of the European-Hematology-Association, JUN 09-12, 2016, Copenhagen, DENMARK
Available from: 2016-08-23 Created: 2016-08-23 Last updated: 2017-11-28Bibliographically approved
Raykova, D., Koos, B., Asplund, A., Gelleri, M., Ivarsson, Y., Danielson, U. H. & Söderberg, O. (2016). Let There Be Light!. Proteomes, 4(4), Article ID 36.
Open this publication in new window or tab >>Let There Be Light!
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2016 (English)In: Proteomes, ISSN 2227-7382, Vol. 4, no 4, article id 36Article, review/survey (Refereed) Published
Abstract [en]

The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Forster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use.

Keywords
high resolution microscopy, protein-protein interactions, post-translational modifications, FRET, in situ PLA, proxHCR
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-316327 (URN)10.3390/proteomes4040036 (DOI)000392385500002 ()
Funder
EU, FP7, Seventh Framework Programme, 278568Swedish Research Council, 2014-02968, D0571301 , 2012-5092
Available from: 2017-05-16 Created: 2017-05-16 Last updated: 2017-05-16Bibliographically approved
Blokzijl, A., Zieba, A., Hust, M., Schirrmann, T., Helmsing, S., Grannas, K., . . . Landegren, U. (2016). Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens. Molecular & cellular proteomics (online), 15(6), 1848-1856
Open this publication in new window or tab >>Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
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2016 (English)In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 15, no 6, p. 1848-1856Article in journal (Refereed) Published
Abstract [en]

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-299590 (URN)10.1074/mcp.M115.055756 (DOI)000377822900006 ()26929218 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationEU, European Research Council, 222635; 241481; 294409Swedish Research CouncilSwedish Research Council, NT-E0383401 MH-K2013-66x-14436-10-5
Available from: 2016-07-22 Created: 2016-07-22 Last updated: 2017-11-28Bibliographically approved
Raja, E., Tzavlaki, K., Vuilleumier, R., Edlund, K., Kahata, K., Zieba, A., . . . Moustakas, A. (2016). The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling. OncoTarget, 7(2), 1120-1143
Open this publication in new window or tab >>The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling
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2016 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 2, p. 1120-1143Article in journal (Refereed) Published
Abstract [en]

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease.

Keywords
BMP; differentiation; Drosophila; LKB1; lung cancer; Pathology Section
National Category
Clinical Laboratory Medicine Basic Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-278888 (URN)10.18632/oncotarget.6683 (DOI)000369951100005 ()26701726 (PubMedID)
Funder
Swedish Research Council, K2007-66X-14936-04-3Swedish Research Council, K2010-67X-14936-07-03Swedish Research Council, K2013-66X-14936-10-5EU, European Research Council, MRTN-2005-005428
Available from: 2016-02-26 Created: 2016-02-26 Last updated: 2018-02-01Bibliographically approved
Johansson Wensman, J., Leuchowius, K.-J., Yan, J., Berg, A.-L., Bode, L., Ludwig, H., . . . Berg, M. (2016). Visualization of Borna Disease Virus Protein Interactions with Host Proteins using in situ Proximity Ligation Assay. British journal of virology, 3(1), 11-23
Open this publication in new window or tab >>Visualization of Borna Disease Virus Protein Interactions with Host Proteins using in situ Proximity Ligation Assay
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2016 (English)In: British journal of virology, ISSN 2055-6128, Vol. 3, no 1, p. 11-23Article in journal (Refereed) Published
Abstract [en]

Borna disease virus type 1 (BDV) comprises highly conserved neurotropic non-segmented negative strand RNA-virus variants causing neurological and behavioral disorders in a wide range of mammalian animals, possibly including humans. Viral persistence in the brain has been frequently observed, however, the exact mechanisms behind BDV’s ability to establish persistence despite a prominent immune response are not known. Here we have used in situ proximity ligation assay (in situ PLA), a selective tool for studying virus-host protein-protein interactions. BDV P (phosphoprotein) and N (nucleoprotein) have previously been reported to interact with several host proteins, thereby interfering with various signaling pathways. In this study, we focused on some of these interactions (BDV P-HMGB1, BDV N/P-Cdc2). First, we used rat glioma cell cultures persistently infected with a laboratory strain of BDV (C6BV) to establish the assay. Next, in situ PLA was applied to detect BDV P in brain tissues of infected animals. Finally, protein-protein interactions were visualized in both C6BV and brain tissues of experimentally as well as naturally infected animals (rat and horse, respectively). BDV proteins and their interactions with host proteins could be shown in cell cultures (HMGB1, Cdc2) and in brain tissues of rat (HMGB1, Cdc2) and horse (Cdc2 only) infected with BDV. In this study, we have for the first time directly visualized protein-protein interactions between BDV and its host, and thereby confirmed previous data to demonstrate findings in cell cultures to be applicable also in experimentally and naturally infected animals.

Keywords
Bornavirus, Virus-host interactions, Viral persistence, Cdc2, HMGB1
National Category
Veterinary Science
Identifiers
urn:nbn:se:uu:diva-310333 (URN)10.17582/journal.bjv/2016.3.1.11.23 (DOI)
Available from: 2016-12-14 Created: 2016-12-14 Last updated: 2017-08-16Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-2883-1925

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