TY  - JOUR
A1  - Andronic, Joseph
A1  - Shirakashi, Ryo
A1  - Pickel, Simone U.
A1  - Westerling, Katherine M.
A1  - Klein, Teresa
A1  - Holm, Thorge
A1  - Sauer, Markus
A1  - Sukhorukov, Vladimir L.
T1  - Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy
JF  - PLoS One
N2  - Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol Pino [m/s] and expression/localization of SLC5A3. Pino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), Pino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in Pino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.
KW  - electrolytes
KW  - isotonic
KW  - membrane proteins
KW  - cell membranes
KW  - hypotonic
KW  - hypotonic solutions
KW  - tonicity
KW  - permeability
Y1  - 2015
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126408
VL  - 10
IS  - 3
ER  - 
TY  - JOUR
A1  - Proppert, Sven
A1  - Wolter, Steve
A1  - Holm, Thorge
A1  - Klein, Theresa
A1  - van de Linde, Sebastian
A1  - Sauer, Markus
T1  - Cubic B-spline calibration for 3D super-resolution measurements using astigmatic imaging
JF  - Optics Express
N2  - In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
KW  - three-dimensional microscopy
KW  - fluorescence microscopy
KW  - medical and biological imaging
KW  - superresolution
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119730
SN  - 1094-4087
VL  - 22
IS  - 9
ER  -