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Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging
KTH, School of Engineering Sciences (SCI), Applied Physics.ORCID iD: 0000-0003-3669-9848
KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5584-9170
KTH, School of Engineering Sciences (SCI), Applied Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.ORCID iD: 0000-0003-0578-4003
(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 [en]
SMLM, PALM, STORM, fusion protein, overexpression
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-215073OAI: oai:DiVA.org:kth-215073DiVA: diva2:1146008
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
In thesis
1. Quantitative bioimaging in single cell signaling
Open this publication in new window or tab >>Quantitative bioimaging in single cell signaling
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. 52 p.
Series
TRITA-FYS, ISSN 0280-316X ; 64
Keyword
Super resolution imaging, fluoresence, bioimaging, cells, FRET, cluster analysis, labeling, image analysis.
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-215076 (URN)978-91-7729-546-4 (ISBN)
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

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