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Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases.

In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products.

Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals.

The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation.

In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases.

Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1202
Keyword [en]
cell signaling, Wnt, Hippo, TGFB
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-281716ISBN: 978-91-554-9529-9 (print)OAI: oai:DiVA.org:uu-281716DiVA: diva2:915351
Public defence
2016-05-20, BMC, B41, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-04-28 Created: 2016-03-29 Last updated: 2016-05-12
List of papers
1. Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
Open this publication in new window or tab >>Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, 12317:1-10 p., 12317Article in journal (Refereed) Published
National Category
Medical Image Processing
Research subject
Computerized Image Processing
Identifiers
urn:nbn:se:uu:diva-260286 (URN)10.1038/srep12317 (DOI)000358358900001 ()26202090 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 259796Swedish Research Council
Available from: 2015-07-23 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
2. Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
Open this publication in new window or tab >>Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
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2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 21, 3407-3415 p.Article in journal (Refereed) Published
Abstract [en]

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-268434 (URN)10.1016/j.jmb.2015.04.015 (DOI)000363823200006 ()25937570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568Swedish Research Council
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
3. Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
Open this publication in new window or tab >>Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
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2015 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 212, no 6, 833-843 p.Article in journal (Refereed) Published
Abstract [en]

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)000355569300001 ()25987724 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), b2011080Swedish Cancer SocietySwedish Research CouncilNIH (National Institute of Health), CA81554; CA081554EU, European Research Council, 259796EU, FP7, Seventh Framework Programme, 306242
Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2017-11-30Bibliographically approved
4. Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
Open this publication in new window or tab >>Crosstalk between WNT and Hippo signaling pathways is affected by cell density and loss of or mutated E-cadherin protein, associated to HDGC.
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-281714 (URN)
Available from: 2016-03-29 Created: 2016-03-29 Last updated: 2016-05-12

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