TY  - JOUR
A1  - Dietz, Mariana S.
A1  - Hasse, Daniel
A1  - Ferraris, Davide M.
A1  - Göhler, Antonia
A1  - Niemann, Hartmut H.
A1  - Heilemann, Mike
T1  - Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells
JF  - BMC Biophysics
N2  - Background: The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. 
Results: To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. 
Conclusions: Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.
KW  - single-molecule photobleaching
KW  - fluorescence correlation spectroscopy
KW  - fluorescence
KW  - EGF receptor
KW  - rat hepatocytes
KW  - structural insights
KW  - Scatter factor
KW  - SEMA domain
KW  - hepatocyte-growth-factor
KW  - invasion protein-INLB
KW  - listeria-monocytogenes
KW  - tyrosine kinase
KW  - living cells
KW  - dimerization
KW  - MET receptor
KW  - Signal transduction
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121835
SN  - 2046-1682
VL  - 6
IS  - 6
ER  - 
TY  - JOUR
A1  - Groeneweg, Femke L.
A1  - van Royen, Martin E.
A1  - Fenz, Susanne
A1  - Keizer, Veer I. P.
A1  - Geverts, Bart
A1  - Prins, Jurrien
A1  - de Kloet, E. Ron
A1  - Houtsmuller, Adriaan B.
A1  - Schmidt, Thomas S.
A1  - Schaaf, Marcel J. M.
T1  - Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP
JF  - PLOS ONE
N2  - Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.
KW  - NF-KAPPA-B
KW  - image correlation spectroscopy
KW  - human mineralocorticoid receptor
KW  - nuclear-pore complexes
KW  - in-vivo
KW  - living cells
KW  - mobility
KW  - transcription
KW  - protein
KW  - reveals
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117085
VL  - 9
IS  - 3
ER  -