TY  - JOUR
A1  - Feldbauer, Katrin
A1  - Schlegel, Jan
A1  - Weissbecker, Juliane
A1  - Sauer, Frank
A1  - Wood, Phillip G.
A1  - Bamberg, Ernst
A1  - Terpitz, Ulrich
T1  - Optochemokine Tandem for Light-Control of Intracellular Ca\(^{2+}\)
JF  - PLoS ONE
N2  - An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca\(^{2+}\)-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca\(^{2+}\) by tandem endosomes into the cytosol via CatCh was visualized using the Ca\(^{2+}\)-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca\(^{2+}\) in response to light.
KW  - capacitance
KW  - endosomes
KW  - cell membranes
KW  - membrane proteins
KW  - intracellular membranes
KW  - vesicles
KW  - confocal laser microscopy
KW  - cytosol
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178921
VL  - 11
IS  - 10
ER  - 
TY  - JOUR
A1  - Djuzenova, Cholpon S.
A1  - Memmel, Simon
A1  - Sukhorukov, Vladimir L.
A1  - Höring, Marcus
A1  - Westerling, Katherine
A1  - Fiedler, Vanessa
A1  - Katzer, Astrid
A1  - Krohne, Georg
A1  - Flentje, Michael
T1  - Cell Surface Area and  Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status
N2  - Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
KW  - cell membranes
KW  - hypotonic
KW  - capacitance
KW  - isotonic
KW  - microvilli
KW  - membrane characteristics
KW  - membrane proteins
KW  - scanning electron microscopy
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111322
ER  -