Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP(3) and Ca(2+) have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca(2+) dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca(2+) alone from the FRET changes evoked by both ligands together. A mutant IP(3)R containing a single amino acid substitution at K508, which is critical for IP(3) binding, did not exhibit this bell-shaped Ca(2+) dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca(2+) sensor, resulted in ∼10-fold reduction in the Ca(2+) dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP(3)R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca(2+) binding affinity, as the mechanism underlying the IP(3)-dependent regulation of the bell-shaped relationship between the IP(3)R activity and cytosolic Ca(2+).

Morphological basis of the lung adenocarcinoma subtypes.

Lung adenocarcinoma (LUAD), which accounts for a large proportion of lung cancers, is divided into five major subtypes based on histologic characteristics. The clinical characteristics, prognosis, and responses to treatments vary among subtypes. Here, we demonstrate that the variations of cell-cell contact energy result in the LUAD subtype-specific morphogenesis. We reproduced the morphologies of the papillary LUAD subtypes with the cellular Potts Model (CPM). Simulations and experimental validations revealed modifications of cell-cell contact energy changed the morphology from a papillary-like structure to micropapillary or solid subtype-like structures. Remarkably, differential gene expression analysis revealed subtype-specific expressions of genes relating to cell adhesion. Knockdown experiments of the micropapillary upregulated ITGA11 gene resulted in the morphological changes of the spheroids produced from an LUAD cell line PC9. This work shows the consequences of gene mutations and gene expressions on patient prognosis through differences in tissue composing physical forces among LUAD subtypes.

Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells.

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 µm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells.

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model.

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.

G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1,4,5-triphosphate receptor-mediated Ca2+ signal regulation.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated calcium (Ca(2+)) release channel and plays important roles in regulation of numerous Ca(2+)-dependent cellular responses. Many intracellular modulators and IP(3)R-binding proteins regulate the IP(3)R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP(3)R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP(3)R. The interaction was favored by the cytosolic Ca(2+) concentration and it functionally inhibited IP(3)R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP(3)R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP(3)R-mediated Ca(2+) signal through a direct interaction with IP(3)R in a cytosolic Ca(2+)-dependent manner.

Inositol 1,4,5-Trisphosphate Receptor Type 3 Regulates Neuronal Growth Cone Sensitivity to Guidance Signals.

During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP3) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP3-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca2+ signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP3 receptor type 3 is deficient. These data suggest a subtype-specific IP3 receptor function in sensitivity adjustment during axon navigation.

Dual-FRET imaging of IP3 and Ca2+ revealed Ca2+-induced IP3 production maintains long lasting Ca2+ oscillations in fertilized mouse eggs.

In most species, fertilization induces Ca2+ transients in the egg. In mammals, the Ca2+ rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP3 generated by PLCζ induces Ca2+ release from the intracellular Ca2+ store through IP3 receptor, termed IP3-induced Ca2+ release. Here, we developed new fluorescent IP3 sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 µM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca2+ and IP3, using IRIS-2s as the IP3 sensor, we developed a new single fluorophore Ca2+ sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP3 concentration ([IP3]) starts to increase before the onset of the first Ca2+ wave. [IP3] stayed at the elevated level with small peaks followed after Ca2+ spikes through Ca2+ oscillations. We detected delays in the peak of [IP3] compared to the peak of each Ca2+ spike, suggesting that Ca2+-induced regenerative IP3 production through PLC produces small [IP3] rises to maintain [IP3] over the basal level, which results in long lasting Ca2+ oscillations in fertilized eggs.

WNT Takes Two to Tango: Molecular Links between the Circadian Clock and the Cell Cycle in Adult Stem Cells.

Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidirectional communication, resulting in circadian clock-gated cell division cycles in species ranging from cyanobacteria to mammals. The identified mechanisms for this phenomenon have expanded beyond intracellular molecular coupling components to include intercellular connections. However, detailed molecular mechanisms, dynamics, and physiological functions of the circadian clock and cell cycle as coupled oscillators remain largely unknown. In this review, we discuss current understanding of this connection in light of recent findings that have uncovered intercellular coupling between the circadian clock in Paneth cells and the cell cycle in intestinal stem cells via WNT signaling. This extends the impact of circadian rhythms regulating the timing of cell divisions beyond the intracellular domain of homogenous cell populations into dynamic, multicellular systems. In-depth understanding of the molecular links and dynamics of these two oscillators will identify potential targets and temporal regimens for effective chronotherapy.

Synthetic Gene Network with Positive Feedback Loop Amplifies Cellulase Gene Expression in Neurospora crassa.

Second-generation or lignocellulosic biofuels are a tangible source of renewable energy, which is critical to combat climate change by reducing the carbon footprint. Filamentous fungi secrete cellulose-degrading enzymes called cellulases, which are used for production of lignocellulosic biofuels. However, inefficient production of cellulases is a major obstacle for industrial-scale production of second-generation biofuels. We used computational simulations to design and implement synthetic positive feedback loops to increase gene expression of a key transcription factor, CLR-2, that activates a large number of cellulases in a filamentous fungus, Neurospora crassa. Overexpression of CLR-2 reveals previously unappreciated roles of CLR-2 in lignocellulosic gene network, which enabled simultaneous induction of approximately 50% of 78 lignocellulosic degradation-related genes in our engineered Neurospora strains. This engineering results in dramatically increased cellulase activity due to cooperative orchestration of multiple enzymes involved in the cellulose degradation pathway. Our work provides a proof of principle in utilizing mathematical modeling and synthetic biology to improve the efficiency of cellulase synthesis for second-generation biofuel production.

Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells.

BACKGROUND: Pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, have a great potential for regenerative medicine. Induced pluripotent stem cells, in particular, are suitable for replacement of tissue by autologous transplantation. However, tumorigenicity is a major risk in clinical application of both embryonic stem cells and induced pluripotent stem cells. This study explores the possibility of manipulating the cell cycle for inhibition of tumorigenicity. METHODS: We genetically modified mouse induced pluripotent stem cells (miPSCs) to overexpress p27 tumor suppressor and examined their proliferation rate, gene expression, cardiac differentiation, tumorigenicity, and therapeutic potential in a mouse model of coronary artery ligation. RESULTS: Overexpression of p27 inhibited cell division of miPSCs, and that inhibition was dependent on the expression level of p27. p27 overexpressing miPSCs had pluripotency characteristics but lost stemness earlier than normal miPSCs during embryoid body and teratoma formation. These cellular characteristics led to none or smaller teratoma when the cells were injected into nude mice. Transplantation of both miPSCs and p27 overexpressing miPSCs into the infarcted mouse heart reduced the infarction size and improved left ventricular function. CONCLUSIONS: The overexpression of p27 attenuated tumorigenicity by reducing proliferation and earlier loss of stemness of miPSCs. The overexpression of p27 did not affect pluripotency and differentiation characteristics of miPSC. Therefore, regulation of the proliferation rate of miPSCs offers great therapeutic potential for repair of the injured myocardium.

Efficient gene editing in Neurospora crassa with CRISPR technology.

BACKGROUND: Efficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, Neurospora crassa. FINDINGS: In this report, we demonstrate efficient gene replacement in a model filamentous fungus, Neurospora crassa, with the CRISPR/Cas9 system. We utilize Cas9 endonuclease and single crRNA:tracrRNA chimeric guide RNA (gRNA) to: (1) replace the endogenous promoter of clr-2 with the ß-tubulin promoter, and (2) introduce a codon optimized fire fly luciferase under the control of the gsy-1 promoter at the csr-1 locus. CLR-2 is one of the core transcription factors that regulate the expression of cellulases, and GSY-1 regulates the conversion of glucose into glycogen. We show that the ß-tubulin promoter driven clr-2 strain shows increased expression of cellulases, and gsy-1-luciferase reporter strain can be easily screened with a bioluminescence assay. CONCLUSION: CRISPR/Cas9 system works efficiently in Neurospora crassa, which may be adapted to Neurospora natural isolates and other filamentous fungi. It will be beneficial for the filamentous fungal research community to take advantage of CRISPR/Cas9 tool kits that enable genetic perturbations including gene replacement and insertions.

Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid.

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration.

Seizure-mediated neuronal activation induces DREAM gene expression in the mouse brain.

Various transcriptional activators are induced in neurons concomitantly with long-lasting neural activity, whereas only a few transcription factors are known to act as neural activity-inducible transcription repressors. In this study, mRNA of DREAM (DRE-antagonizing modulator), a Ca(2+)-modulated transcriptional repressor, was demonstrated to accumulate in the mouse brain after pentylenetetrazol (PTZ)-induced seizures. Accumulation in the mouse hippocampus reached maximal level in the late phase (at 7-8 h) after PTZ injection. Kainic acid induced the same response. Interestingly, the late induction of DREAM expression required new protein synthesis and was blocked by MK801 suggesting that Ca(2+)-influx via NMDA receptors is necessary for the PTZ-mediated DREAM expression. In situ hybridization revealed that PTZ-induced DREAM mRNA accumulation was observed particularly in the dentate gyrus, cerebral cortex, and piriform cortex. The results of the present study demonstrate that DREAM is a neural activity-stimulated late gene and suggest its involvement in adaptation to long-lasting neuronal activity.

Seizure-mediated accumulation of the beta subunit of Ca2+/calmodulin-dependent protein kinase II in nuclei of mouse brain cells.

We identified a 45-kDa protein by 2D electrophoresis that was enhanced following pentylenetetrazol (PTZ)-mediated seizures. Mass-spectrography of this protein revealed the beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIbeta), although no evidence for increase in bulk CaMKIIbeta transcripts was obtained. Physicochemical parameters of the 45-kDa species coincided with those of the type 7 isoform of CaMKIIbeta, CaMKIIbeta7. Reverse transcription-polymerase chain reaction revealed the existence of the CaMKIIbeta7 transcript in the mouse brain, but its RNA content was small and was not elevated by PTZ injection. CaMKIIbeta7 protein is thought to be accumulated in the nuclei of brain cells by PTZ-mediated seizure via some cellular mechanisms other than transcriptional and post-transcriptional regulation.

Phospholipase C-ß1 and ß4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+) signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+) oscillations in terms of the time constant of Ca(2+) spike amplitude decay and the Ca(2+) oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+) signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+) signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-ß1, -ß3, -ß4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-ß1 and PLC-ß4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-ß1 and PLC-ß4 resulted in specific changes in the characteristics of Ca(2+) oscillations, such as the time constant of the temporal changes in the Ca(2+) spike amplitude and the Ca(2+) oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+) release, can cause cell-to-cell variability in the patterns of Ca(2+) signals and that PLC-ß1 and PLC-ß4 contribute to generate cell-specific Ca(2+) signals evoked by G protein-coupled receptor stimulation.