Cell-based optimization and characterization of genetically encoded location-based biosensors for Cdc42 or Rac activity.

Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium.

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

Not So Dry After All: DRY Mutants of the AT1A Receptor and H1 Receptor Can Induce G-Protein-Dependent Signaling.

G-protein-coupled receptors (GPCRs) are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize the functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here, we show that both H1R DRY mutant and the AT1AR DRY mutant are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to the common belief, they do not constitute suitable tools for the dissection of the arrestin-mediated, G protein-independent signaling downstream of these receptors.

Superfolder mTurquoise2ox optimized for the bacterial periplasm allows high efficiency in vivo FRET of cell division antibiotic targets.

Fluorescent proteins (FPs) are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non-native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging and Förster resonance energy transfer (FRET). To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise2ox (sfTq2ox ) and is able to withstand challenging, oxidizing environments. sfTq2ox has improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide bonds. The improved sfTq2ox has the same spectroscopic properties as mTq2, that is, high fluorescence lifetime and quantum yield. The sfTq2ox -mNeongreen FRET pair allows the detection of periplasmic protein-protein interactions with energy transfer rates exceeding 40%. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way for in vivo antibiotic screening. SIGNIFICANCE: The periplasmic space of Gram-negative bacteria contains many regulatory, transport and cell wall-maintaining proteins. A preferred method to investigate these proteins in vivo is by the detection of fluorescent protein fusions. This is challenging since most fluorescent proteins do not fluoresce in the oxidative environment of the periplasm. We assayed popular fluorescent proteins for periplasmic functionality and describe key factors responsible for periplasmic fluorescence. Using this knowledge, we engineered superfolder mTurquoise2ox (sfTq2ox ), a new cyan fluorescent protein, capable of bright fluorescence in the periplasm. We show that our improvements come without a trade-off from its parent mTurquoise2. Employing sfTq2ox as FRET donor, we show the direct in vivo interaction and disruption of unique periplasmic antibiotic targets FtsB and FtsL.