Morphotype-specific calcium signaling in human microglia.

Background: Key functions of Ca signaling in rodent microglia include monitoring the brain state as well as the surrounding neuronal activity and sensing the danger or damage in their vicinity. Microglial Ca dyshomeostasis is a disease hallmark in many mouse models of neurological disorders but the Ca signal properties of human microglia remain unknown.

Methods: We developed a novel genetically-encoded ratiometric Ca indicator, targeting microglial cells in the freshly resected human tissue, organotypically cultured tissue slices and analyzed in situ ongoing Ca signaling of decades-old microglia dwelling in their native microenvironment.

Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca influx into the -Golgi Network (TGN) promotes secretory cargo sorting by the Ca-ATPase SPCA1 and the luminal Ca binding protein Cab45. Cab45 oligomerizes upon local Ca influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca concentration.

Probing the interstitial calcium compartment.

Calcium in interstitial fluids is a crucial ion pool for entry into cells through a plethora of calcium-permeable channels. It is also sensed actively by dedicated receptors. While the mechanisms of global calcium homeostasis and regulation in body fluids appear well understood, more efforts and new technology are needed to elucidate local calcium handling in the small and relatively isolated interstitial spaces between cells.

CRISPR/Cas9-based directed evolution in mammalian cells.

An increasingly powerful set of new CRISPR/Cas-based methods is becoming available for directed evolution of proteins in mammalian cells. Although in vitro techniques or microbial expression systems have been dominating directed evolution, there are now promising approaches to diversify proteins in mammalian cells in situ. This can be achieved by simple indel mutagenesis or more sophisticated homology repair mechanisms for cassette mutagenesis of coding sequences.

Targeted In Situ Protein Diversification and Intra-organelle Validation in Mammalian Cells.

Engineered proteins must be phenotypically selected for function in the appropriate physiological context. Here, we present a versatile approach that allows generating panels of mammalian cells that express diversified heterologous protein libraries in the cytosol or subcellular compartments under stable conditions and in a single-variant-per-cell manner. To this end we adapt CRISPR/Cas9 editing technology to diversify targeted stretches of a protein of interest in situ.

Calcium-sensing receptor regulates intestinal dipeptide absorption via Ca signaling and IK activation.

Although absorption of di- and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 (PEPT1, also called solute carrier family 15 member 1 (SLC15A1)), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca ([Ca ] ), (b) whether the calcium sensing receptor (CaSR) is involved in dipeptide-elicited [Ca ] signaling, and (c) what potential consequences of [Ca ] signaling may enhance enterocyte dipeptide absorption. Dipeptide Gly-Sar and CaSR agonist spermine markedly raised [Ca ] in villous enterocytes, which was abolished by NPS-2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor.

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators.

Functional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies.

Dynamic tuning of FRET in a green fluorescent protein biosensor.

Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with -dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.

Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles†.

In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles.

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum.

The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum.

Calcium Influx through Plasma-Membrane Nanoruptures Drives Axon Degeneration in a Model of Multiple Sclerosis.

Axon loss determines persistent disability in multiple sclerosis patients. Here, we use in vivo calcium imaging in a multiple sclerosis model to show that cytoplasmic calcium levels determine the choice between axon loss and survival. We rule out the endoplasmic reticulum, glutamate excitotoxicity, and the reversal of the sodium-calcium exchanger as sources of intra-axonal calcium accumulation and instead identify nanoscale ruptures of the axonal plasma membrane as the critical path of calcium entry.

Imaging-Based Screening Platform Assists Protein Engineering.

Protein engineering involves generating and screening large numbers of variants for desired properties. While modern DNA technology has made it easy to create protein diversity on the DNA level, the selection and validation of candidate proteins from large libraries remains a challenge. We built a screening platform that integrates high-quality fluorescence-based image analysis and robotic picking of bacterial colonies.

Color Processing in the Early Visual System of Drosophila.

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits.

Brain tumour cells interconnect to a functional and resistant network.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions.

Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels.

Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior.

FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.

Background: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step.

New Method: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM).

In vivo odourant response properties of migrating adult-born neurons in the mouse olfactory bulb.

Juxtaglomerular neurons (JGNs) of the mammalian olfactory bulb are generated throughout life. Their integration into the preexisting neural network, their differentiation and survival therein depend on sensory activity, but when and how these adult-born cells acquire responsiveness to sensory stimuli remains unknown. In vivo two-photon imaging of retrovirally labelled adult-born JGNs reveals that ~90% of the cells arrive at the glomerular layer after day post injection (DPI) 7.

A recoverable state of axon injury persists for hours after spinal cord contusion in vivo.

Therapeutic strategies for spinal cord injury (SCI) commonly focus on regenerating disconnected axons. An alternative approach would be to maintain continuity of damaged axons, especially after contusion. The viability of such neuropreservative strategies depends on the degree to which initially injured axons can recover.

Putting a finishing touch on GECIs.

More than a decade ago genetically encoded calcium indicators (GECIs) entered the stage as new promising tools to image calcium dynamics and neuronal activity in living tissues and designated cell types in vivo. From a variety of initial designs two have emerged as promising prototypes for further optimization: FRET (Förster Resonance Energy Transfer)-based sensors and single fluorophore sensors of the GCaMP family. Recent efforts in structural analysis, engineering and screening have broken important performance thresholds in the latest generation for both classes.

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice.

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking.

Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo.

Mitochondrial redox signals have a central role in neuronal physiology and disease. Here we describe a new optical approach to measure fast redox signals with single-organelle resolution in living mice that express genetically encoded redox biosensors in their neuronal mitochondria. Moreover, we demonstrate how parallel measurements with several biosensors can integrate these redox signals into a comprehensive characterization of mitochondrial function.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C.

Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator.

To study T cell activation in vivo in real time, we introduced a newly developed fluorescence resonance energy transfer-based, genetically encoded calcium indicator into autoantigen-specific and non-autoantigen-specific CD4(+) T cells. Using two-photon microscopy, we explored the responses of retrovirally transduced calcium indicator-expressing T cells to antigen in the lymph nodes and the central nervous system. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around antigen-presenting cells and an instant rise of cytosolic calcium.