HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: fj.06-6280comv1 21/3/700    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wotzlaw, C. Articles by Fandrey, J. Search for Related Content PubMed PubMed Citation Articles by Wotzlaw, C. Articles by Fandrey, J.

Optical analysis of the HIF-1 complex in living cells by FRET and FRAP

Christoph Wotzlaw^*, Teresa Otto^*, Utta Berchner-Pfannschmidt^*, Eric Metzen, Helmut Acker^* and Joachim Fandrey^*,1

^* Institut für Physiologie, Universität Duisburg-Essen, Essen, Germany; and

Institut für Physiologie, Universität zu Lübeck, Lübeck, Germany

¹Correspondence: Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail: joachim.fandrey{at}uni-due.de

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1 and its constitutive ß subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophore-labeled HIF-1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1 than of HIF-1ß, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between and ß subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models.—Wotzlaw, C., Otto, T., Berchner-Pfannschmidt, U., Metzen, E., Acker, H., Fandrey, J. Optical analysis of the HIF-1 complex in living cells by FRET and FRAP.

Key Words: fluorescence resonance energy transfer • fluorescence recovery after photobleaching • hypoxia-inducible factor-1 • oxygen sensing