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The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.

BACKGROUND:Choroid plexus epithelial cells express high levels of transthyretin, produce cerebrospinal fluid and many of its proteins, and make up the blood-cerebrospinal fluid barrier. Choroid plexus epithelial cells are vital to brain health and may be involved in neurological diseases. Transgenic mice containing fluorescent and luminescent reporters of these cells would facilitate their study in health and disease, but prior transgenic reporters lost expression over the early postnatal period. METHODS:Human bacterial artificial chromosomes in which the transthyretin coding sequence was replaced with DNA for tdTomato or luciferase 2 were used in pronuclear injections to produce transgenic mice. These mice were characterized by visualizing red fluorescence, immunostaining, real-time reverse transcription polymerase chain reaction, and luciferase enzyme assay. RESULTS:Reporters were faithfully expressed in cells that express transthyretin constitutively, including choroid plexus epithelial cells, retinal pigment epithelium, pancreatic islets, and liver. Expression of tdTomato in choroid plexus began at the appropriate embryonic age, being detectable by E11.5. Relative levels of tdTomato transcript in the liver and choroid plexus paralleled relative levels of transcripts for transthyretin. Expression remained robust over the first postnatal year, although choroid plexus transcripts of tdTomato declined slightly with age whereas transthyretin remained constant. TdTomato expression patterns were consistent across three founder lines, displayed no sex differences, and were stable across several generations. Two of the tdTomato lines were bred to homozygosity, and homozygous mice are healthy and fertile. The usefulness of tdTomato reporters in visualizing and analyzing live Transwell cultures was demonstrated. Luciferase activity was very high in homogenates of choroid plexus and continued to be expressed through adulthood. Luciferase also was detectable in eye and pancreas. CONCLUSIONS:Transgenic mice bearing fluorescent and luminescent reporters of transthyretin should prove useful for tracking transplanted choroid plexus epithelial cells, for purifying the cells, and for reporting their derivation from stem cells. They also should prove useful for studying transthyretin synthesis by other cell types, as transthyretin has been implicated in many functions and conditions, including clearance of β-amyloid peptides associated with Alzheimer's disease, heat shock in neurons, processing of neuropeptides, nerve regeneration, astrocyte metabolism, and transthyretin amyloidosis.

The lack of efficient tools to image non-repetitive genes in living cells has limited our ability to explore the functional impact of the spatiotemporal dynamics of such genes. Here, we addressed this issue by developing a CRISPR-Tag system using one to four highly active sgRNAs to specifically label protein-coding genes with a high signal-to-noise ratio for visualization by wide-field fluorescence microscopy. Our approach involves assembling a CRISPR-Tag within the intron region of a fluorescent protein and then integrating this cassette to N- or C-terminus of a specific gene, which enables simultaneous real-time imaging of protein and DNA of human protein-coding genes, such as HIST2H2BE, LMNA and HSPA8 in living cells. This CRISPR-Tag system, with a minimal size of ~250 bp DNA tag, represents an easily and broadly applicable technique to study the spatiotemporal organization of genomic elements in living cells.

Mechanical forces between cells and extracellular matrix (ECM) influence cell shape and function. Tendons are ECM-rich tissues connecting muscles with bones that bear extreme tensional force. Analysis of transgenic zebrafish expressing mCherry driven by the tendon determinant scleraxis reveals that tendon fibroblasts (tenocytes) extend arrays of microtubule-rich projections at the onset of muscle contraction. In the trunk, these form a dense curtain along the myotendinous junctions at somite boundaries, perpendicular to myofibers, suggesting a role as force sensors to control ECM production and tendon strength. Paralysis or destabilization of microtubules reduces projection length and surrounding ECM, both of which are rescued by muscle stimulation. Paralysis also reduces SMAD3 phosphorylation in tenocytes and chemical inhibition of TGFβ signaling shortens tenocyte projections. These results suggest that TGFβ, released in response to force, acts on tenocytes to alter their morphology and ECM production, revealing a feedback mechanism by which tendons adapt to tension.

Microglia are the resident immune cell of the central nervous system (CNS), and serve to protect and maintain the local brain environment. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R); administration of CSF1R inhibitors that cross the blood brain barrier (BBB) lead to the elimination of up to 99% of microglia, depending on CNS exposure and treatment duration. Once microglia are depleted, withdrawal of inhibitor stimulates repopulation of the entire CNS with new cells, conceivably enabling a therapeutic strategy for beneficial renewal of the entire microglial tissue. We have explored the kinetics and limits of this repopulation event and show that the rate of microglial repopulation is proportional to the extent of microglial depletion - greater depletion of microglia results in more rapid repopulation. Using a CSF1R inhibitor formulation that eliminates approximately 99% of microglia within 7 days, we subjected mice to multiple rounds of elimination (7 days' treatment) and repopulation (7 days' recovery) and found that the brain only has the capacity for a single complete repopulation event; subsequent elimination and CSF1R inhibitor withdrawal fail to repopulate the brain. However, if the recovery time between, or after, cycles is extended sufficiently then the brain can ultimately repopulate. These kinetic studies define the opportunities and possible limits of the remarkable renewal capacities of microglia.

Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.

Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally nonfluorescent at acidic conditions due to the decrease of anionic chromophores upon protonation, but are fluorescent at physiological pH, creating interfering fluorescence from nonvesicle regions. Here we developed a novel acid-brightening fluorescent protein (abFP) that fluoresces strongly at acidic pH but is nonfluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on the mammalian cell surface. We further demonstrated that abFP-tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP-tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery.

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( KD < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.

Autism spectrum disorders (ASD) are a set of complex neurodevelopmental disorders for which there is currently no targeted therapeutic approach. It is thought that alterations of genes regulating migration and synapse formation during development affect neural circuit formation and result in aberrant connectivity within distinct circuits that underlie abnormal behaviors. However, it is unknown whether deviant developmental trajectories are circuit-specific for a given autism risk-gene. We used MRI to probe changes in functional and structural connectivity from childhood to adulthood in Fragile-X (Fmr1-/y) and contactin-associated (CNTNAP2-/-) knockout mice. Young Fmr1-/y mice (30 days postnatal) presented with a robust hypoconnectivity phenotype in corticocortico and corticostriatal circuits in areas associated with sensory information processing, which was maintained until adulthood. Conversely, only small differences in hippocampal and striatal areas were present during early postnatal development in CNTNAP2-/- mice, while major connectivity deficits in prefrontal and limbic pathways developed between adolescence and adulthood. These findings are supported by viral tracing and electron micrograph approaches and define 2 clearly distinct connectivity endophenotypes within the autism spectrum. We conclude that the genetic background of ASD strongly influences which circuits are most affected, the nature of the phenotype, and the developmental time course of the associated changes.

Synonymous codon choice can have dramatic effects on ribosome speed and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs. Here, we have modeled this variation in translation elongation by using a feed-forward neural network to predict the ribosome density at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and characterized large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of translation speeds predicted with our model. Levels of the fluorescent protein in budding yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo; that this information can be harnessed to design coding sequences; and that control of translation elongation alone is sufficient to produce large quantitative differences in protein output.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

C57BL/6 mice exhibit spontaneous cerebellar malformations consisting of heterotopic neurons and glia in the molecular layer of the posterior vermis, indicative of neuronal migration defect during cerebellar development. Recognizing that many genetically engineered (GE) mouse lines are produced from C57BL/6 ES cells or backcrossed to this strain, we performed histological analyses and found that cerebellar heterotopia were a common feature present in the majority of GE lines on this background. Furthermore, we identify GE mouse lines that will be valuable in the study of cerebellar malformations including diverse driver, reporter, and optogenetic lines. Finally, we discuss the implications that these data have on the use of C57BL/6 mice and GE mice on this background in studies of cerebellar development or as models of disease.

MreB is a bacterial actin that is important for cell shape and cell wall biosynthesis in many bacterial species. MreB also plays crucial roles in Myxococcus xanthus gliding motility, but the underlying mechanism remains unknown. Here we tracked the dynamics of single MreB particles in M. xanthus using single-particle tracking photoactivated localization microscopy. We found that a subpopulation of MreB particles moves rapidly along helical trajectories, similar to the movements of the MotAB-like gliding motors. The rapid MreB motion was stalled in the mutants that carried truncated gliding motors. Remarkably, M. xanthus MreB moves one to two orders of magnitude faster than its homologs that move along with the cell wall synthesis machinery in Bacillus subtilis and Escherichia coli, and this rapid movement was not affected by the inhibitors of cell wall biosynthesis. Our results show that in M. xanthus, MreB provides a scaffold for the gliding motors while the gliding machinery drives the movement of MreB filaments, analogous to the interdependent movements of myosin motors and actin in eukaryotic cells.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Repetitive and perseverative behaviors are common features of a number of neuropsychiatric diseases such as Angelman's syndrome, Tourette's syndrome, obsessive-compulsive disorder, and autism spectrum disorders. The oxytocin system has been linked to the regulation of repetitive behavior in both animal models and humans, but many of its downstream targets have still to be found. We report that the melanin-concentrating hormone (MCH) system is a target of the oxytocin system in regulating one repetitive behavior, marble burying. First we report that nearly 60% of MCH neurons express oxytocin receptors, and demonstrate using rabies mediated tract tracing that MCH neurons receive direct presynaptic input from oxytocin neurons. Then we show that MCH receptor knockout (MCHR1KO) mice and MCH ablated animals display increased marble burying response while central MCH infusion decreases it. Finally, we demonstrate the downstream role of the MCH system on oxytocin mediated marble burying by showing that central infusions of MCH and oxytocin alone or together reduce it while antagonizing the MCH system blocks oxytocin-mediated reduction of this behavior. Our findings reveal a novel role for the MCH system as a mediator of the role of oxytocin in regulating marble-burying behavior in mice.

BackgroundThe complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain.MethodsTo identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1+ neurons in both wild type mice and a mouse model of Alzheimer's disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis.ResultsWhile C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1+ neurons, the brains of C1qa FL/FL :Cx3cr1 CreERT2 mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 CreERT2/WganJ mice. C1q expression in C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice relative to controls, and C1qa FL/FL : Cx3cr1 CreERT2/WganJ mice had minimal, if any, reduction in plasma C1q.ConclusionsThus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 CreERT2/WganJ deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders.

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc.

ObjectivePrevious genetic lineage tracing studies showed that Sox10+ cells differentiate into vascular mural cells, limited to neural crest-derived blood vessels in craniofacial tissues, aortic arch, pulmonary arch arteries, brachiocephalic, carotid arteries, and thymus. The purpose of this study was to investigate the contribution of Sox10+ cells to the vascular development in other tissues and organs and their relationship with neural crest.Approach and resultsUsing genetic lineage tracing technique based on Cre/LoxP system, we examined blood vessels in the adult organs of the mice expressing Sox10-Cre/Rosa-LoxP-red fluorescent protein or Wnt1-Cre/Rosa-LoxP-red fluorescent protein by immunohistological analysis. In addition to previously reported tissues and organs derived from neural crest, we showed that Sox10+ cells also contributed to vascular mural cells in the lung, spleen, and kidney, which are derived from non-neural crest origin as evidenced by red fluorescent protein-negative blood vessels in these 3 organs of Wnt1-Cre/Rosa-LoxP-red fluorescent protein mice.ConclusionsThis study demonstrates that Sox10+ cells contribute to pericytes and smooth muscle cells in most parts of the body, including those from neural crest and non-neural crest, which has significant implications in vascular remodeling under physiological and pathological conditions.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.

In bacteria various tactic responses are mediated by the same cellular pathway, but sensing of physical stimuli remains poorly understood. Here, we combine an in-vivo analysis of the pathway activity with a microfluidic taxis assay and mathematical modeling to investigate the thermotactic response of Escherichia coli. We show that in the absence of chemical attractants E. coli exhibits a steady thermophilic response, the magnitude of which decreases at higher temperatures. Adaptation of wild-type cells to high levels of chemoattractants sensed by only one of the major chemoreceptors leads to inversion of the thermotactic response at intermediate temperatures and bidirectional cell accumulation in a thermal gradient. A mathematical model can explain this behavior based on the saturation-dependent kinetics of adaptive receptor methylation. Lastly, we find that the preferred accumulation temperature corresponds to optimal growth in the presence of the chemoattractant serine, pointing to a physiological relevance of the observed thermotactic behavior.

The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in ∼65-75% of alveolar epithelial type II (ATII) cells, but in only ∼45-50% of the integrin β4+ population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in ∼50% of ATII cells and ∼80% of integrin β4+ cells. Upon dox treatment of adults, deletion in ATII cells and integrin β4+ cells in SP-C-tTg mice dropped significantly to ∼20% and ∼6%, respectively, whereas CCSP-tTg mice deleted in ∼57% of ATII and ∼40% of integrin β4+ cells. Interestingly, untreated CCSP-tTg mice also deleted in ∼40% of integrin β4+ cells, indicating significant leakiness of CCSP-tTg in β4+ cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.

Nkx2-5 is a homeobox-containing transcriptional regulator that serves as one of the earliest markers of cardiac lineage commitment. To study the role of Nkx2-5-expressing progenitors at specific time points in cardiac development, we have generated a novel and inducible NKX2-5 mouse line by knocking in a CreER cassette into the Nkx2-5 genomic locus, while preserving the endogenous Nkx2-5 gene to avoid haploinsufficiency. We evaluated the specificity and efficiency of CreER activity after 4-OHT injection by crossing Nkx2-5CreER/+ mice with a Rosa26tdT/+ reporter strain. Our immunohistochemistry results confirmed Cre-induced tdTomato expression specifically in cells expressing Nkx2-5. These cells were mainly cardiomyocytes and were observed in the embryonic heart as early as day 9.5. Additionally, quantitative polymerase chain reaction on postnatal hearts showed enriched expression of Nkx2-5 in isolated tdTomato-expressing cells. No tdTomato expression was observed in Nkx2-5CreER/+ ;Rosa26tdT/+ mice in the absence of 4-OHT, confirming the inducible nature of CreER activity. The Nkx2-5/CreER mouse model described in this article will serve as an invaluable tool to trace myocardial lineage and to temporally induce genetic manipulation in a selective population of cardiac progenitors during embryonic development and in the adult heart.

Brightness analysis of fluorescence fluctuation experiments has been used to successfully measure the oligomeric state of proteins at the plasma membrane, in the nucleoplasm, and in the cytoplasm of living cells. Here we extend brightness analysis to the nuclear envelope (NE), a double membrane barrier separating the cytoplasm from the nucleoplasm. Results obtained by applying conventional brightness analysis to fluorescently tagged proteins within the NE exhibited an unusual concentration dependence. Similarly, the autocorrelation function of the fluorescence fluctuations exhibited unexpected changes with protein concentration. These observations motivated the application of mean-segmented Q analysis, which identified the existence of a fluctuation process distinct from molecular diffusion in the NE. We propose that small changes in the separation of the inner and outer nuclear membrane are responsible for the additional fluctuation process, as suggested by results obtained for luminal and nuclear membrane-associated EGFP-tagged proteins. Finally, we applied these insights to study the oligomerization of the luminal domains of two nuclear membrane proteins, nesprin-2 and SUN2, which interact transluminally to form a nuclear envelope-spanning linker molecular bridge known as the linker of the nucleoskeleton and cytoskeleton complex.

There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

Natural genetic promoters are regulated by multiple cis and trans regulatory factors. For quantitative studies of these promoters, the concentration of only a single factor is typically varied to obtain the dose response or transfer function of the promoters with respect to the factor. Such design of experiments has limited our ability to understand quantitative, combinatorial interactions between multiple regulatory factors at promoters. This limitation is primarily due to the intractable number of experimental combinations that arise from multifactorial design of experiments. To overcome this major limitation, we integrate impact printing and cell-free systems to enable multi-dimensional studies of genetic promoters. We first present a gradient printing system which comprises parallel piezoelectric cantilever beams as a scalable actuator array to generate droplets with tunable volumes in the range of 100 pL-10 nL, which facilitates highly accurate direct dilutions in the range of 1-10 000-fold in a 1 μL drop. Next, we apply this technology to study interactions between three regulatory factors at a synthetic genetic promoter. Finally, a mathematical model of gene regulatory modules is established using the multi-parametric and multi-dimensional data. Our work creates a new frontier in the use of cell-free systems and droplet printing for multi-dimensional studies of synthetic genetic constructs.

Gastric leiomyosarcoma is a recalcitrant cancer and the chemotherapy strategy is controversial. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of gastric leiomyosarcoma to identify an effective therapeutic regimen to develop individualized precision medicine for this disease. The gastric leiomyosarcoma obtained from a patient was first grown in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP) to stably label the tumor stroma. The RFP-expressing tumor was then passaged orthotopically in the gastric wall of non-transgenic nude mice to establish an imageable PDOX (iPDOX) model. The bright fluorescent tumor was readily imaged over time to determine drug efficacy. Four weeks after implantation, 70 PDOX nude mice were divided into 7 groups: control without treatment (n = 10); doxorubicin (DOX) (2.4 mg/kg, intraperitoneally (i.p.), once a week for 2 weeks, n = 10); gemcitabine (GEM)/ docetaxel (DOC) (GEM: 100 mg/kg, DOC: 20 mg/kg, i.p., once a week for 2 weeks, n = 10); cyclophosphamide (CPA) (140 mg/kg, i.p., once a week for 2 weeks, n = 10); temozolomide (TEM) (25 mg/kg, orally, daily for 14 consecutive days, n = 10); yondelis (YON) (0.15 mg/kg, i.v., once a week for 2 weeks, n = 10); pazopanib (PAZ) (100 mg/kg, orally, daily for 14 consecutive days, n = 10). On day 14 from initiation of treatment, all treatments except PAZ significantly inhibited tumor growth compared with untreated control (DOX: p < 0.01, GEM/DOC: p < 0.01, CPA: p < 0.01, TEM: p < 0.01, YON: p < 0.01) on day 14 after initiation. In addition, only GEM/DOC was more significantly effective than DOX (p < 0.05). GEM/DOC could regress the leimyosarcoma in the PDOX model and has important clinical potential for precision individual treatment of leiomyosarcoma patients.

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.

Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

Medial ganglionic eminence (MGE) transplantation rescues disease phenotypes in various preclinical models with interneuron deficiency or dysfunction, including epilepsy. While underlying mechanism(s) remains unclear to date, a simple explanation is that appropriate synaptic integration of MGE-derived interneurons elevates GABA-mediated inhibition and modifies the firing activity of excitatory neurons in the host brain. However, given the complexity of interneurons and potential for transplant-derived interneurons to integrate or alter the host network in unexpected ways, it remains unexplored whether synaptic connections formed by transplant-derived interneurons safely mirror those associated with endogenous interneurons. Here, we combined optogenetics, interneuron-specific Cre driver mouse lines, and electrophysiology to study synaptic integration of MGE progenitors. We demonstrated that MGE-derived interneurons, when transplanted into the hippocampus of neonatal mice, migrate in the host brain, differentiate to mature inhibitory interneurons, and form appropriate synaptic connections with native pyramidal neurons. Endogenous and transplant-derived MGE progenitors preferentially formed inhibitory synaptic connections onto pyramidal neurons but not endogenous interneurons. These findings demonstrate that transplanted MGE progenitors functionally integrate into the postnatal hippocampal network.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

Voltage-sensing dyes and voltage-sensing fluorescence proteins have been continually improved and as a result have provided a wealth of insights into neuronal circuits. Further improvements in voltage-sensing dyes and voltage-sensing fluorescence proteins are needed, however, for routine detection of single action potentials across a large number of individual neurons in a large field-of-view of a live mammalian brain. On the other hand, recent experiments and calculations suggest that semiconducting nanoparticles could act as efficient voltage sensors, suitable for the above-mentioned task. This study presents quantum mechanical calculations, including Auger recombination rates, of the quantum-confined Stark effect in membrane-embedded semiconducting nanoparticles, examines their possible utility as membrane voltage sensors, and provide design rules for their structure and composition.

Our laboratory pioneered patient-derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient-like, in contrast to the ectopic subcutaneous-transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft-tissue sarcoma (UPS-STS) PDOX model acquired bright RFP-expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non-colored nude mice was non-invasively imageable. A PDOX nude mouse model of UPS-STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently passaged to RFP transgenic mice, and after tumor growth were then passaged to non-transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS-STS PDOX models. UPS-STS PDOX tumors, previously grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non-transgenic nude mice maintained the bright fluorescence and were non-invasively imageable. FV1000 confocal imaging revealed diffusely distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non-transgenic mice. These results demonstrate a powerful method to make the PDOX UPS-STS model brightly fluorescent for non-invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP-labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma-targeting drugs, whereby the stromal cells are a visual target. J. Cell. Biochem. 118: 361-365, 2017. © 2016 Wiley Periodicals, Inc.