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Quantitative bioimaging in single cell signaling
KTH, School of Engineering Sciences (SCI), Applied Physics.ORCID iD: 0000-0003-3669-9848
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.

Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.

Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.

In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.

Abstract [sv]

Avbildning av biologiska prover har i flera hundra år varit ett sätt för forskare att undersöka biologiska system. Med utvecklingen av immunofluoresens inmärkn-ing och fluoresens-mikroskopi förbättrades de viktigaste aspekterna av mikroskopi,kontrast och specificitet. Sedan 1941 har vi sett kontinuerligt mer mångsidigt och frekvent användning av fluorosense-mikroskopi i biologisk forskning.

Jon-mikroskopi har länge varit en metod att studera signalering i cell-system. Genom användning av fluorosenta jon-sensorer går det att mäta variationer avjon koncentrationer i levande celler som resultat av yttre påverkan. Genom att använda Ca2+ mikroskopi har jag visat att det finns en omvänd koppling mellan kalcium-kanaler i plasma-membran och angiotensin II typ 1 receptorn (ett proteininvolverat i blodtrycksreglering). Detta har direkta implikationer för behandlingav högt blodtryck, en av de mer vanliga sjukdomarna i västvärlden idag med överen miljard drabbade patienter i världen 2016.

Efter detta projekt vidgades mitt fokus till att inkludera superupplösnings-mikroskopi. Denna avhandling inkluderar ett arbete fokuserat på tolkningen av superupplösnings-mikroskopi data från neuronal Na+, K+ - ATPase α3, en jon-pump som återställer cellernas jonbalans i samband med cell signalering. Mikroskopi-datan korreleras mot elektrofysiologi experiment och modeller för att illustrera möjliga artefakter i superupplösnings-mikroskopi som måste tas i beaktande i samband med tolkning av data.

Jag fortsatte med att utveckla mjukvara för analys av data från singel-molekyl-lokalisations-mikroskopi där fokuset för mjukvaran framförallt varit på användarvänligheten. Detta då jag hoppas att den kommer vara användbar för ett bredare forskingsfält. Mjukvaran användes även i ett separat projekt för att identifiera överuttrycks-artefakter i transfekterade celler.

I det avslutande arbetet använder jag superupplösnings-mikroskopi för att karakterisera de tidiga stegen i mitokondriell apoptos. Jag identifierar när och var i cellen de olika proteinerna involverade i apoptos signaleringen är aktiverade och interagerar.

Place, publisher, year, edition, pages
Kungliga Tekniska högskolan, 2017. , p. 52
Series
TRITA-FYS, ISSN 0280-316X ; 64
Keyword [en]
Super resolution imaging, fluoresence, bioimaging, cells, FRET, cluster analysis, labeling, image analysis.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-215076ISBN: 978-91-7729-546-4 (print)OAI: oai:DiVA.org:kth-215076DiVA, id: diva2:1146013
Public defence
2017-10-27, Air and Fire, Science for Life Laboratory, Tomtebodavägen 23a, Solna, 09:00 (English)
Opponent
Supervisors
Note

QC 20171003

Available from: 2017-10-03 Created: 2017-10-01 Last updated: 2017-10-03Bibliographically approved
List of papers
1. AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
Open this publication in new window or tab >>AT(1)-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers
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2017 (English)In: BMC Cardiovascular Disorders, ISSN 1471-2261, E-ISSN 1471-2261, Vol. 17, no 1, article id 126Article in journal (Refereed) Published
Abstract [en]

Background: Blockers of angiotensin II type 1   receptor (AT 1 R) and the voltage gated calcium channel 1.2 (Ca V 1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT 1 R signaling and whether there is a reciprocal regulation of AT 1 R signaling by Ca V 1.2.

Methods: To elucidate these questions, we have studied the Ca 2+  signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca 2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of Ca V 1.2 blockers.  Semi- quantitative imaging methods were used to assess the plasma membrane expression of AT 1 R and G-protein activation.

Results: Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca 2+  response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca 2+  response. The up-regulation of the Ca 2+  response to repeated 1 nM AngII doses and the down- egulation of the Ca 2+  response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT 1 R plasma membrane expression. The Ca 2+  response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the Ca V 1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT 1 R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT 1 R is sensitive to calcium influx.

Conclusions: Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca 2+  channel blockers as mono-therapy in hypertension. 

Place, publisher, year, edition, pages
Stockholm: BioMed Central, 2017
Keyword
Calcium, AT1R, Cell imaging, VGCC, hypertension
National Category
Medical and Health Sciences
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-203989 (URN)10.1186/s12872-017-0562-x (DOI)000401701400001 ()28514967 (PubMedID)2-s2.0-85019540748 (Scopus ID)
Funder
Swedish Heart Lung FoundationSwedish Research CouncilMagnus Bergvall FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170328

Available from: 2017-03-21 Created: 2017-03-21 Last updated: 2017-11-29Bibliographically approved
2. Sodium pump organization in dendritic spines
Open this publication in new window or tab >>Sodium pump organization in dendritic spines
2016 (English)In: NEUROPHOTONICS, ISSN 2329-423X, Vol. 3, no 4, article id 041803Article in journal (Refereed) Published
Abstract [en]

Advancement in fluorescence imaging with the invention of several super-resolution microscopy modalities (e.g., PALM/STORM and STED) has opened up the possibility of deciphering molecular distributions on the nanoscale. In our quest to better elucidate postsynaptic protein distribution in dendritic spines, we have applied these nanoscopy methods, where generated results could help improve our understanding of neuronal functions. In particular, we have investigated the principal energy transformer in the brain, i.e., the Na+; K+-ATPase (or sodium pump), an essential protein responsible for maintaining resting membrane potential and a major controller of intracellular ion homeostasis. In these investigations, we have focused on estimates of protein amount, giving assessments of how variations may depend on labeling strategies, sample analysis, and choice of nanoscopic imaging method, concluding that all can be critical factors for quantification. We present a comparison of these results and discuss the influences this may have for homeostatic sodium regulation in neurons and energy consumption.

Place, publisher, year, edition, pages
SPIE - International Society for Optical Engineering, 2016
Keyword
dendritic spine, sodium pump, photoactivated localization microscopy, stimulated emission depletion microscopy
National Category
Neurosciences Neurology
Identifiers
urn:nbn:se:kth:diva-194264 (URN)10.1117/1.NPh.3.4.041803 (DOI)000384427300002 ()27175374 (PubMedID)2-s2.0-84979072971 (Scopus ID)
Note

QC 20161024

Available from: 2016-10-24 Created: 2016-10-21 Last updated: 2018-01-14Bibliographically approved
3. SMLocalizer, a GPU accelerated ImageJ plugin for single molecule localization microscopy
Open this publication in new window or tab >>SMLocalizer, a GPU accelerated ImageJ plugin for single molecule localization microscopy
2018 (English)In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 34, no 1, p. 137-Article in journal (Refereed) Published
Abstract [en]

SMLocalizer combines the availability of ImageJ with the power of GPU processing for fast and accurate analysis of single molecule localization microscopy data. Analysis of 2D and 3D data in multiple channels is supported.

Place, publisher, year, edition, pages
Oxford University Press, 2018
Keyword
Single-molecule localization microscopy, CUDA, PALM, STORM
National Category
Software Engineering
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-215066 (URN)10.1093/bioinformatics/btx553 (DOI)000419591100027 ()2-s2.0-85040058231 (Scopus ID)
Funder
Swedish Research Council, VR 2015-04198Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20171003

Available from: 2017-10-01 Created: 2017-10-01 Last updated: 2018-01-29Bibliographically approved
4. Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging
Open this publication in new window or tab >>Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the ability to quantitatively monitor endogenous and exogenous protein expression competition on the single molecule level. Through incorporation of an N-terminal hemagglutinin (HA) epitope to amMaple3 fused Na,K-ATPase (α1 isoform), using PALM and STORM imaging we investigatethe increase in plasma membrane density at the cost of competitive expression. Quantification of plasma membrane protein density revealed a time dependent increase over time of totalprotein content. Results show that plasma membrane densities increased by more than 60%,comparing 17h and 41h transfection times, whilst endogenous levels were simultaneously reduced by 20 %.

Keyword
SMLM, PALM, STORM, fusion protein, overexpression
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-215073 (URN)
Funder
Swedish Research Council, VR 2013-6041Swedish Research Council, 2015-04198Swedish Foundation for Strategic Research
Note

QC 20171003

Available from: 2017-10-01 Created: 2017-10-01 Last updated: 2017-10-03Bibliographically approved
5. Experimental validation of predicted cancer genes using FRET
Open this publication in new window or tab >>Experimental validation of predicted cancer genes using FRET
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Huge amounts of data are generated in genome wide experiments, designed to investigatediseases with complex genetic causes. Follow up of all potential leads produced by suchexperiments is currently cost prohibitive and time consuming. Gene prioritization toolsalleviate these constraints by directing further experimental efforts towards the mostpromising candidate targets. Recently a gene prioritization tool called MaxLink was shown tooutperform other widely used state-of-the-art prioritization tools in a large scale in silicobenchmark. An experimental validation of predictions made by MaxLink has however beenlacking. In this study we used Fluorescent Resonance Energy Transfer, an establishedexperimental technique for detection of protein-protein interactions, to validate potentialcancer genes predicted by MaxLink. Our results provide confidence in the use of MaxLink forselection of new targets in the battle with polygenic diseases.

Keyword
FRET, protein interaction, high throughput
National Category
Bioinformatics and Systems Biology Biophysics
Identifiers
urn:nbn:se:kth:diva-215074 (URN)
Note

QC 20171003

Available from: 2017-10-01 Created: 2017-10-01 Last updated: 2017-10-03Bibliographically approved
6. Super resolution imaging reveals details in hyperglycemic induced apoptosis in kidney cells
Open this publication in new window or tab >>Super resolution imaging reveals details in hyperglycemic induced apoptosis in kidney cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The role of the Bcl-family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods in studies of isolated mitochondria and transfected cell lines. There is however little knowledge about the mechanisms for Bcl protein interaction leading to apoptosis in intact cells. In particular, the time sequence and location for Bcl protein interaction has so far only been described in hypothetical models.Here we have used Stimulated Emission Depletion (STED) microscopy and Single Molecule Localization Microscopy (SMLM) to study the apoptotic process in immune-stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl-2 proteins, we used the nearest-neighbor algorithm. The anti-apoptotic protein Bcl-xLl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis-inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl-xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl-xL interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol1. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl-xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non-specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return.

Keyword
Super-resolution, STED, SMLM, diabetes
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-215075 (URN)
Note

QC 20171003

Available from: 2017-10-01 Created: 2017-10-01 Last updated: 2017-10-03Bibliographically approved

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