Postsynaptic competition between calcineurin and PKA regulates mammalian sleep-wake cycles.

The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep-wake cycle in mammals. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration.

Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells.

Epidermal growth factor receptor ligands (EGFRLs) consist of seven proteins. In stark contrast to the amassed knowledge concerning the epidermal growth factor receptors themselves, the extracellular dynamics of individual EGFRLs remain elusive. Here, employing fluorescent probes and a tool for triggering ectodomain shedding of EGFRLs, we show that EREG, a low-affinity EGFRL, exhibits the most rapid and efficient activation of EGFR in confluent epithelial cells and mouse epidermis.

Circadian ribosome profiling reveals a role for the upstream open reading frame in sleep.

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance.

Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro.

Distinct phosphorylation states of mammalian CaMKIIβ control the induction and maintenance of sleep.

The reduced sleep duration previously observed in Camk2b knockout mice revealed a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII)β as a sleep-promoting kinase. However, the underlying mechanism by which CaMKIIβ supports sleep regulation is largely unknown. Here, we demonstrate that activation or inhibition of CaMKIIβ can increase or decrease sleep duration in mice by almost 2-fold, supporting the role of CaMKIIβ as a core sleep regulator in mammals.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter.

Two-photon AMPK and ATP imaging reveals the bias between rods and cones in glycolysis utility.

In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed.

A human isogenic iPSC-derived cell line panel identifies major regulators of aberrant astrocyte proliferation in Down syndrome.

Astrocytes exert adverse effects on the brains of individuals with Down syndrome (DS). Although a neurogenic-to-gliogenic shift in the fate-specification step has been reported, the mechanisms and key regulators underlying the accelerated proliferation of astrocyte precursor cells (APCs) in DS remain elusive. Here, we established a human isogenic cell line panel based on DS-specific induced pluripotent stem cells, the XIST-mediated transcriptional silencing system in trisomic chromosome 21, and genome/chromosome-editing technologies to eliminate phenotypic fluctuations caused by genetic variation.

Visualization of Spatially-Controlled Vasospasm by Sympathetic Nerve-Mediated ROCK Activation.

Contraction of vascular smooth muscle is regulated primarily by calcium concentration and secondarily by ROCK activity within the cells. In contrast to the wealth of information regarding regulation of calcium concentration, little is known about the spatiotemporal regulation of ROCK activity in live blood vessels. Here, we report ROCK activation in subcutaneous arterioles in a transgenic mouse line that expresses a genetically encoded ROCK biosensor based on the principle of Fӧrster resonance energy transfer by two-photon excitation in vivo imaging.

Gsx2 is required for specification of neurons in the inferior olivary nuclei from Ptf1a-expressing neural progenitors in zebrafish.

Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene In this study, we found that the homeobox gene was co-expressed with in IO progenitors in zebrafish. Both and zebrafish mutants showed a strong reduction or loss of IO neurons.

Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins.

Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells.

The regulatory landscape of the Dlx gene system in branchial arches: Shared characteristics among Dlx bigene clusters and evolution.

The mammalian Dlx genes encode homeobox-type transcription factors and are physically organized as convergent bigene clusters. The paired Dlx genes share tissue specificity in the expression profile. Genetic regulatory mechanisms, such as intergenic enhancer sharing between paired Dlx genes, have been proposed to explain this conservation of bigene structure.

Booster, a Red-Shifted Genetically Encoded Förster Resonance Energy Transfer (FRET) Biosensor Compatible with Cyan Fluorescent Protein/Yellow Fluorescent Protein-Based FRET Biosensors and Blue Light-Responsive Optogenetic Tools.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light.

Mark1 regulates distal airspace expansion through type I pneumocyte flattening in lung development.

During the later stages of lung development, two types of pneumocytes, cuboidal type II (AECII) and flattened type I (AECI) alveolar epithelial cells, form distal lung saccules. Here, we highlight how fibroblasts expressing MAP-microtubule affinity regulating kinase 1 (Mark1) are required for the terminal stages of pulmonary development, called lung sacculation. In Mark1-knockout (KO) mice, distal sacculation and AECI flattening are significantly impaired.

FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics.

Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2.

Next-generation human genetics for organism-level systems biology.

Systems-biological approaches, such as comprehensive identification and analysis of system components and networks, are necessary to understand design principles of human physiology and pathology. Although reverse genetics using mouse models have been used previously, it is a low throughput method because of the need for repetitive crossing to produce mice having all cells of the body with knock-out or knock-in mutations. Moreover, there are often issues from the interspecific gap between humans and mice.

Expression of plasma membrane calcium ATPases confers Ca/H exchange in rodent synaptic vesicles.

Ca transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca transporter which is active at neutral pH and the other is mediated by a low affinity Ca/H antiporter which is maximally active at alkaline pH of 8.

Easy and efficient production of completely embryonic-stem-cell-derived mice using a micro-aggregation device.

There is an increasing demand for genetically modified mice produced without crossing, for rapid phenotypic screening studies at the organismal level. For this purpose, generation of completely embryonic-stem-cell (ESC)-derived chimeric mice without crossing is now possible using a microinjection or aggregation method with 3i culture medium. However, the microinjection of ESCs into blastocyst, morula, or 8-cell-stage embryos requires a highly skilled operator.

Muscarinic Acetylcholine Receptors Chrm1 and Chrm3 Are Essential for REM Sleep.

Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e.

A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging.

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine.

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs.

A Highly Sensitive FRET Biosensor for AMPK Exhibits Heterogeneous AMPK Responses among Cells and Organs.

AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, is a potential target for type 2 diabetes. Although extensive in vitro studies have revealed the complex regulation of AMPK, much remains unknown about the regulation in vivo. We therefore developed transgenic mice expressing a highly sensitive fluorescence resonance energy transfer (FRET)-based biosensor for AMPK, called AMPKAR-EV.