ACVR1 p.Q207E causes classic fibrodysplasia ossificans progressiva and is functionally distinct from the engineered constitutively active ACVR1 p.Q207D variant.

Fibrodysplasia ossificans progressiva (FOP) is a disabling genetic disorder of progressive heterotopic ossification (HO). Here, we report a patient with an ultra-rare point mutation [c.619C>G, p.Q207E] located in a codon adjacent to the most common FOP mutation [c.617G>A, p.R206H] of Activin A Receptor, type 1 (ACVR1) and that affects the same intracellular amino acid position in the GS activation domain as the engineered constitutively active (c.a.) variant p.Q207D. It was predicted that both mutations at residue 207 have similar functional effects by introducing a negative charge. Transgenic p.Q207D-c.a. mice have served as a model for FOP HO in several in vivo studies. However, we found that the engineered ACVR1(Q207D-c.a.) is significantly more active than the classic FOP mutation ACVR1(R206H) when overexpressed in chicken limbs and in differentiation assays of chondrogenesis, osteogenesis and myogenesis. Importantly, our studies reveal that the ACVR1(Q207E) resembles the classic FOP receptor in these assays, not the engineered ACVR1(Q207D-c.a.). Notably, reporter gene assays revealed that both naturally occurring FOP receptors (ACVR1(R206H) and ACVR1(Q207E)) were activated by BMP7 and were sensitive to deletion of the ligand binding domain, whereas the engineered ACVR1(Q207D-c.a.) exhibited ligand independent activity. We performed an in silico analysis and propose a structural model for p.Q207D-c.a. that irreversibly relocates the GS domain into an activating position, where it becomes ligand independent. We conclude that the engineered p.Q207D-c.a. mutation has severe limitations as a model for FOP, whereas the naturally occurring mutations p.R206H and p.Q207E facilitate receptor activation, albeit in a reversible manner.

Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization.

Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH4(+) uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane.

Optical oxygen micro- and nanosensors for plant applications.

Pioneered by Clark's microelectrode more than half a century ago, there has been substantial interest in developing new, miniaturized optical methods to detect molecular oxygen inside cells. While extensively used for animal tissue measurements, applications of intracellular optical oxygen biosensors are still scarce in plant science. A critical aspect is the strong autofluorescence of the green plant tissue that interferes with optical signals of commonly used oxygen probes. A recently developed dual-frequency phase modulation technique can overcome this limitation, offering new perspectives for plant research. This review gives an overview on the latest optical sensing techniques and methods based on phosphorescence quenching in diverse tissues and discusses the potential pitfalls for applications in plants. The most promising oxygen sensitive probes are reviewed plus different oxygen sensing structures ranging from micro-optodes to soluble nanoparticles. Moreover, the applicability of using heterologously expressed oxygen binding proteins and fluorescent proteins to determine changes in the cellular oxygen concentration are discussed as potential non-invasive cellular oxygen reporters.

Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

Single-fluorophore membrane transport activity sensors with dual-emission read-out.

We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer is important for the successful generation of single-fluorophore-based dual-emission sensors.

Fluorescent sensors reporting the activity of ammonium transceptors in live cells.

Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors ('AmTrac' and 'MepTrac'). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track. DOI:http://dx.doi.org/10.7554/eLife.00800.001.

The fibrodysplasia ossificans progressiva R206H ACVR1 mutation activates BMP-independent chondrogenesis and zebrafish embryo ventralization.

Patients with classic fibrodysplasia ossificans progressiva, a disorder characterized by extensive extraskeletal endochondral bone formation, share a recurrent mutation (R206H) within the glycine/serine-rich domain of ACVR1/ALK2, a bone morphogenetic protein type I receptor. Through a series of in vitro assays using several mammalian cell lines and chick limb bud micromass cultures, we determined that mutant R206H ACVR1 activated BMP signaling in the absence of BMP ligand and mediated BMP-independent chondrogenesis that was enhanced by BMP. We further investigated the interaction of mutant R206H ACVR1 with FKBP1A, a glycine/serine domain-binding protein that prevents leaky BMP type I receptor activation in the absence of ligand. The mutant protein exhibited reduced binding to FKBP1A in COS-7 simian kidney cell line assays, suggesting that increased BMP pathway activity in COS-7 cells with R206H ACVR1 is due, at least in part, to decreased binding of this inhibitory factor. Consistent with these findings, in vivo analyses of zebrafish embryos showed BMP-independent hyperactivation of BMP signaling in response to the R206H mutant, resulting in increased embryonic ventralization. These data support the conclusion that the mutant R206H ACVR1 receptor in FOP patients is an activating mutation that induces BMP signaling in a BMP-independent and BMP-responsive manner to promote chondrogenesis, consistent with the ectopic endochondral bone formation in these patients.