Adjobo-Hermans, M. J. W. | Search bioRxiv

bioRxiv2022

SMDT1 variants impair EMRE-mediated mitochondrial calcium uptake in patients with muscle involvement

Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments provide evidence that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of the oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the patients with SMDT1 variants result from aberrant mitochondrial Ca2+ uptake.

genetics↗

bioRxiv2017

A FRET based biosensor for measuring Gα13 activation in single cells

Forster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human G13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged G13 subunits were used as FRET donor and combined with cp173Venus fused to the G{gamma}2 subunit as the acceptor. We constructed G13 biosensors by generating a single plasmid that produces G13-mTurquoise2, G{beta}1 and cp173Venus-G{gamma}2. The G13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the G13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the G13 biosensor is a valuable tool for live-cell measurements that probe G13 activation.

cell biology↗