TY  - JOUR
A1  - Kuhlemann, Alexander
A1  - Beliu, Gerti
A1  - Janzen, Dieter
A1  - Petrini, Enrica Maria
A1  - Taban, Danush
A1  - Helmerich, Dominic A.
A1  - Doose, Sören
A1  - Bruno, Martina
A1  - Barberis, Andrea
A1  - Villmann, Carmen
A1  - Sauer, Markus
A1  - Werner, Christian
T1  - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
JF  - Frontiers in Synaptic Neuroscience
N2  - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
KW  - super-resolution microscopy (SRM)
KW  - click-chemistry
KW  - dSTORM
KW  - GABA-A receptor
KW  - genetic code expansion
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035
SN  - 1663-3563
VL  - 13
ER  - 
TY  - JOUR
A1  - Börtlein, Charlene
A1  - Draeger, Annette
A1  - Schoenauer, Roman
A1  - Kuhlemann, Alexander
A1  - Sauer, Markus
A1  - Schneider-Schaulies, Sybille
A1  - Avota, Elita
T1  - The neutral sphingomyelinase 2 is required to polarize and sustain T Cell receptor signaling
JF  - Frontiers in Immunology
N2  - By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca\(^{2+}\) mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.
KW  - neutral sphingomyelinase 2
KW  - T cells
KW  - ceramides
KW  - PKCζ,
KW  - the microtubule-organizing center
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176572
VL  - 9
IS  - 815
ER  -