Biology subjects

Obata, Y.

Publications and source records attributed to Obata, Y..

11 recordsLinked to original sources

bioRxiv2025

A Novel Cell-Type Specific Circadian Reporter Mouse Reveals Self-Sustained Food Entrainable Nature in Enteric Neurons

Global luciferase reporter gene technology is an important real-time readout for the analysis of circadian oscillations. However, because nearly all cells in the body possess cell-autonomous circadian oscillators, developing cell-type-specific reporter systems is essential to dissect how these oscillators interact within complex multicellular tissues and are modulated by brain-body circadian signals. The intestine is a complex organ composed of diverse cell types with distinct origins and functions, and it exhibits robust daily rhythms in physiological activities driven by intrinsic circadian clocks. While the circadian regulation of the mucosal compartment, including the intestinal epithelium, has been relatively well characterized, the mechanisms governing the circadian rhythms in the cells of the muscularis externa remain largely unexplored. Here, we report a novel Cre-dependent Per2-luciferase reporter mouse that enables precise measurement of circadian oscillations in specific peripheral cell populations ex vivo and demonstrate its utility in revealing the hierarchical circadian chrono-architecture within the intestine. Ex vivo gut explants from mice expressing the reporter in one of five major cell types of the muscularis externa--enteric neurons (ENs), enteric glial cells (EGCs), interstitial cells of Cajal (ICCs), smooth muscle cells (SMCs), and muscularis macrophages (MMs)--exhibited robust, self-sustained circadian bioluminescence rhythms, indicating that all of these cell types possess cell-autonomous circadian oscillators. Notably, ENs in the small intestine entrained more rapidly to feeding schedules than did those in the colon, revealing regional differences in food entrainment among the gut clocks. Moreover, the clocks of ENs, SMCs, and MMs shifted their phase in response to daytime-restricted feeding, whereas ICC clocks remained unaffected even after three weeks of restricted feeding. These findings demonstrate that distinct intestinal cell types possess unique entrainment properties and that feeding rhythms can induce heterogeneous phase shifts within the gut clocks. Given that circadian disruption, such as that caused by shift work, contributes to intestinal disorders, this reporter system could provide a powerful platform to dissect the mechanisms linking intercellular circadian desynchrony to intestinal homeostasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/688705v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@11250fforg.highwire.dtl.DTLVardef@18397f2org.highwire.dtl.DTLVardef@6ccd24org.highwire.dtl.DTLVardef@10fffef_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA cell-type-specific circadian reporter mouse enables dissection of the chronoarchitecture of complex multicellular organs (e.g., the intestine) C_LIO_LIAll five major intestinal cell types contain autonomous circadian oscillators peaking at night C_LIO_LIEnteric neurons in small intestine entrain to feeding cycles faster than that in colon C_LIO_LIInterstitial cells of Cajal in both the small intestine and colon do not entrain to feeding cycles C_LIO_LIChemogenetic activation of enteric neurons mimics food entrainment, shifting their circadian phase. C_LI

neuroscience↗

bioRxiv2025

Disruption of Marrow Microenvironments in Chronic Lymphocytic Leukemia by High-Resolution Synchrotron Micro-Computed Tomography

Chronic lymphocytic leukemia (CLL) is associated with increased fracture risk unexplained by standard bone density scans, suggesting underlying microstructural alterations. To investigate this, we used high-resolution synchrotron micro-computed tomography (SR{micro}CT) on bone marrow biopsies from 17 CLL patients, who were stratified into low and high infiltration groups using objective, data-driven clustering. To our knowledge, this is the first report to quantify these changes. High CLL marrow infiltration was associated with a 40.3% reduction in lacuna density and a 101% increase in the normalized adipose surface area-to-volume ratio, a metric indicating greater structural fragmentation. Both changes correlated with leukemic infiltration percentage and showed partial reversal after therapy in a longitudinal case. Furthermore, high CLL burden significantly altered the morphological distributions of the remaining lacunar osteocyte (p < 0.001). We identify a novel marrow remodeling phenotype in CLL characterized by osteocyte depletion and adipose disruption. These changes likely contribute to skeletal fragility and represent potential microstructural biomarkers for assessing marrow health more accurately than conventional imaging.

physiology↗

bioRxiv2025

Golgi retention of KIT in gastrointestinal stromal tumour cells is phospholipase D activity-dependent

A constitutively active mutant of the receptor protein-tyrosine kinase KIT is a major cause of gastrointestinal stromal tumours (GISTs). Recently, we discovered that during biosynthetic transport, the KIT mutant (KITmut) is retained in the Golgi/trans-Golgi network (TGN), where it activates downstream molecules. This retention is dependent on the phospholipase C{gamma}2-protein kinase D2-PI4 kinase III{beta} (PLC{gamma}2-PKD2-PI4KIII{beta}) pathway, which KITmut activates at the Golgi/TGN. The activated cascade aberrantly recruits GGA1 and the {gamma}-adaptin subunit of AP1, resulting in KITmut retention in the Golgi/TGN. However, the precise mechanisms, including the mediators and effectors in the pathway, remain unclear. In humans, the phosphatidic acid-generating enzymes, phospholipase D1 (PLD1) and PLD2, are known downstream proteins of PKD. In the presence of the PLD inhibitor CAY10594, KITmut is released from the Golgi/TGN and subsequently degraded in lysosomes, leading to signal inactivation. Knockdown experiments indicated that PLD2 plays a role in KITmut retention. KITmut activates PLD2 through PKD2, but not PI4KIII{beta}, for Golgi/TGN retention. PLD activity is required for the association of {gamma}-adaptin with GGA1. Therefore, the KIT-PLC{gamma}2-PKD2 pathway separately activates PLD2 and PI4KIII{beta} to recruit {gamma}-adaptin and GGA1. Collectively, these results suggest that KITmut retention is dependent on the activation of the PLC{gamma}2-PKD2-PLD2 cascade in GIST.

cell biology↗

bioRxiv2023

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, which ARFs among AFR family proteins are inhibited by BFA or M-COPA, that is, which ARFs are involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

cell biology↗

bioRxiv2023

Whole-bone toughness is linked to canal and osteocyte lacunae deficits in the ZDSD type 2 diabetic rat model

Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk independent of bone mass. The exact origin of this increased fracture risk is still not well understood. Using a polygenic diabetic rat model, synchrotron radiation micro-computed tomography (SRCT), and in situ scanning electron microscope (SEM) fracture toughness, we related the changes at the microscale to toughness and material properties of diabetic rat femurs. The diabetic rat model (ZDSD) displayed overnight fasting hyperglycemia and an increased AGEs content. Additionally, we measured the impairment of post-yield properties and toughness in diabetic rats. The cortical geometry and porosity were also affected in this ZDSD model. We measured a decrease in osteocyte lacunar density associated with a decreased lacunar volume. Moreover, we found decreased canal density while maintaining a similar canal diameter. These results indicate that diabetes impairs bone remodeling, affecting bone microstructure. Because canals and lacunae are also linked with extrinsic toughening mechanisms, we attribute the decreased toughness largely to these microstructural changes. In conclusion, we showed that changes in lacunae and canal density, combined with AGEs accumulation, decreased toughness in T2DM rat bone.

bioengineering↗

bioRxiv2022

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in GIST cells

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes KITs Golgi retention. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT is unable to trigger downstream activation. In the Golgi area, KIT activates the PKD2-phosphatidylinositol 4-kinaseIII{beta} (PKD2-PI4KIII{beta}) pathway through phospholipase {gamma}2 (PLC{gamma}2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in KIT release from the Golgi region, indicating that these PKD2-related pathways are responsible for the Golgi retention of KIT. Our findings unveil the molecular mechanisms underlying KIT mislocalization and provide evidence for a new strategy for inhibition of oncogenic signaling.

cancer biology↗

bioRxiv2022

Macrophages regulate gastrointestinal motility through complement component 1q

Peristaltic movement of the intestine propels food down the length of the gastrointestinal tract to promote nutrient absorption. Interactions between intestinal macrophages and the enteric nervous system regulate gastrointestinal motility, yet we have an incomplete understanding of the molecular mediators of this crosstalk. Here we identify complement component 1q (C1q) as a macrophage product that regulates gut motility. Macrophages were the predominant source of C1q in the mouse intestine and most extraintestinal tissues. Although C1q mediates complement-mediated killing of bacteria in the bloodstream, we found that C1q was not essential for immune defense of the intestine. Instead, C1q-expressing macrophages were localized to the intestinal submucosal plexus where they closely associated with enteric neurons and expressed surface markers characteristic of nerve-adjacent macrophages in other tissues. Mice with a macrophage-specific deletion of C1qa showed changes in enteric neuronal gene expression, increased peristaltic activity, and accelerated intestinal transit. Our findings identify C1q as a key regulator of gastrointestinal motility and provide enhanced insight into the crosstalk between macrophages and the enteric nervous system.

immunology↗

bioRxiv2021

Luminal nutrients activate distinct patterns in submucosal and myenteric neurons in the mouse small intestine

Nutrient signals sensed by enteroendocrine cells are conveyed to the enteric nervous system (ENS) to initiate intestinal reflexes. We addressed whether there are specific enteric pathways dedicated to detecting different luminal nutrients. Calcium imaging was performed on intact jejunal preparations from Wnt1-cre;R26R-GCaMP3 and Villin-cre;R26R-GCaMP3 mice which express a fluorescent calcium indicator in their ENS or intestinal epithelium, respectively. Glucose, acetate, and L-phenylalanine were perfused onto the mucosa whilst imaging underlying enteric neurons. Nutrient transport or diffusion across the mucosa was mimicked by applying nutrients onto sensory nerve endings in a villus, or onto myenteric ganglia. The nutrients perfused onto the mucosa each elicited Ca2+ transients in submucosal neurons and in distinct patterns of myenteric neurons. Notably, the neurochemical subtypes of myenteric neurons that responded differed between the nutrients, while submucosal responders were predominantly cholinergic. Nutrients applied into villi or onto ganglia did not elicit specific neuronal responses but did stimulate Ca2+ signaling in the mucosal epithelium. These data suggest that nutrients are first detected at the level of the epithelium and that the ENS is capable of discriminating between different compositions of luminal content. Furthermore, our data show that responses to mucosal stimulation are primarily in the myenteric plexus and submucosal neurons respond secondarily.

neuroscience↗

bioRxiv2021

FLT3-ITD transduces autonomous growth signals during its biosynthetic trafficking in acute myelogenous leukemia cells.

FMS-like tyrosine kinase 3 (FLT3) in hematopoietic cells binds to its ligand at the plasma membrane (PM), then transduces growth signals. FLT3 gene alterations that lead the kinase to assume its permanently active form, such as internal tandem duplication (ITD) and D835Y substitution, are found in 30~40% of acute myelogenous leukemia (AML) patients. Thus, the drugs for molecular targeting of FLT3 mutants have been developed for the treatment of AML. Several groups have reported that compared with wild-type FLT3 (FLT3-wt), FLT3 mutants are retained in organelles, resulting in low levels of PM localization of the receptor. However, the precise subcellular localization of mutant FLT3 remains unclear, and the relationship between oncogenic signaling and the mislocalization is not completely understood. In this study, we show that in cell lines established from AML patients, endogenous FLT3-ITD but not FLT3-wt clearly accumulates in the perinuclear region. Our co-immunofluorescence assays demonstrate that Golgi markers are co-localized with the perinuclear region, indicating that FLT3-ITD mainly localizes to the Golgi region in AML cells. FLT3-ITD biosynthetically traffics to the Golgi apparatus and remains there in a manner dependent on its tyrosine kinase activity. A tyrosine kinase inhibitor midostaurin (PKC412) markedly decreases in FLT3-ITD retention and increases in the PM levels of the mutant. FLT3-ITD activates downstream in the endoplasmic reticulum (ER) and the Golgi apparatus during its biosynthetic trafficking. Results of our trafficking inhibitor treatment assays show that FLT3-ITD in the ER activates STAT5, whereas that in the Golgi can cause the activation of AKT and ERK. We provide evidence that FLT3-ITD signals from the early secretory compartments before reaching the PM in AML cells.

cancer biology↗

bioRxiv2020

Enteric glia as a source of neural progenitors in adult zebrafish

The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glia markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease.

neuroscience↗

bioRxiv2019

Neuronal programming by microbiota enables environmental regulation of intestinal motility

Environmental signals modulate the activity of the nervous system and harmonize its output with the outside world. Synaptic activity is crucial for integrating sensory and effector neural pathways but the role of transcriptional mechanisms as environmental sensors in the nervous system remains unclear. By combining a novel strategy for transcriptomic profiling of enteric neurons with microbiota manipulation, we demonstrate that the transcriptional programs of intestinal neural circuits depend on their anatomical and physiological context. We also identify the ligand-dependent transcription factor Aryl hydrocarbon Receptor (AhR) is an intrinsic regulator of enteric nervous system output. AhR is instated as a neuronal biosensor in response to microbiota colonization allowing resident enteric neurons to directly monitor and respond to the intestinal microenvironment. We suggest that AhR signaling integrates neuronal activity with host defence mechanisms towards gut homeostasis and health.\n\nOne Sentence SummaryMicrobiota induce expression of AhR in enteric neurons of the distal intestine enabling them to respond to environmental signals.

neuroscience↗