Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.

Ultraspeed single-molecule tracking with <25-μs resolution and electron tomography show that transmembrane proteins and phospholipids in the plasma membrane hop among submicrometer compartments of the same size, probably delimited by the anchored-transmembrane-protein pickets lining the actin-based membrane-skeleton fence, once every 1–58 ms.

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1-3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an approximately 50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.

Circadian clock oscillation emerges in mouse embryo in the later developmental stages. Although circadian clock development is closely correlated with cellular differentiation, the mechanisms of its emergence during mammalian development are not well understood. Here, we demonstrate an essential role of the posttranscriptional regulation of subsequent to the cellular differentiation for the emergence of circadian clock oscillation in mouse fetal hearts and mouse embryonic stem cells (ESCs). In mouse fetal hearts, no apparent oscillation of cell-autonomous molecular clock was detectable around E10, whereas oscillation was clearly visible in E18 hearts. Temporal RNA-sequencing analysis using mouse fetal hearts reveals many fewer rhythmic genes in E10-12 hearts (63, no core circadian genes) than in E17-19 hearts (483 genes), suggesting the lack of functional circadian transcriptional/translational feedback loops (TTFLs) of core circadian genes in E10 mouse fetal hearts. In both ESCs and E10 embryos, CLOCK protein was absent despite the expression of mRNA, which we showed was due to -dependent translational suppression of CLOCK. The CLOCK protein is required for the discernible molecular oscillation in differentiated cells, and the posttranscriptional regulation of plays a role in setting the timing for the emergence of the circadian clock oscillation during mammalian development.

The circadian clock in mammalian cells is cell-autonomously generated during the cellular differentiation process, but the underlying mechanisms are not understood. Here we show that perturbation of the transcriptional program by constitutive expression of transcription factor c-Myc and DNA methyltransferase 1 (Dnmt1) ablation disrupts the differentiation-coupled emergence of the clock from mouse ESCs. Using these model ESCs, 484 genes are identified by global gene expression analysis as factors correlated with differentiation-coupled circadian clock development. Among them, we find the misregulation of Kpna2 (Importin-α2) during the differentiation of the c-Myc-overexpressed and Dnmt1 −/− ESCs, in which sustained cytoplasmic accumulation of PER proteins is observed. Moreover, constitutive expression of Kpna2 during the differentiation culture of ESCs significantly impairs clock development, and KPNA2 facilitates cytoplasmic localization of PER1/2. These results suggest that the programmed gene expression network regulates the differentiation-coupled circadian clock development in mammalian cells, at least in part via posttranscriptional regulation of clock proteins.T he circadian clock is an intrinsic time-keeping system that regulates essential physiological functions such as sleep/wake cycles, body temperature, and metabolism (1-3). In the mammalian clock system the central pacemaker resides in the suprachiasmatic nucleus of the hypothalamus, coordinating cell-autonomous molecular oscillators throughout the body to perform tissue-specific functions. The molecular oscillator consists of transcriptional/ translational feedback loops of clock genes. Two transcription factors, CLOCK and BMAL1, heterodimerize and transactivate core clock genes such as the Period (Per1, 2, 3), Cryptochrome (Cry1, 2), and Rev-Erbα genes via E-box regulatory elements. PER and CRY proteins, in turn, translocate into the nucleus to suppress CLOCK/BMAL1 activity, leading to cyclic expression of these clock genes (4-9). Furthermore, REV-ERBα negatively regulates Bmal1 transcription via the RORE promoter element, thus driving antiphasic expression patterns of Bmal1 (10, 11).Despite uncovering the emergence of the circadian rhythms that occur during development (12-14), the precise mechanism of circadian clock development in mammalian cells remains unclear. Recently it has been found that pluripotent ES cells (ESCs) do not display discernible circadian molecular oscillations, whereas in vitro-differentiated ESCs displayed robust circadian oscillations of reporter expression (15-17). Moreover, we also have shown that circadian oscillations were abolished when differentiated cells were reprogrammed to regain pluripotency by expression of reprogramming factors (Oct3/4, Sox2, Klf4, and c-Myc) (15). These results suggested that the emergence of the cell-autonomous circadian oscillator is coupled with cellular differentiation. Cellular differentiation, as well as reprogramming, results in global alterations of the transcriptional program and epi...

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1 + CD44 high CD4 T cells as well as CD95 + GL7 + germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders. From the cellular to the organismal levels, circadian clocks regulate various essential biological processes to enable anticipation of and adaptation to the daily environmental changes from Earth rotation 1. Modernization of our society is accompanied by a dramatic change in human lifestyle, with unprecedented increases in, for example, night shift work and nocturnal feeding/recreational activities 2. Recent epidemiological studies have revealed shift workers as being at a higher risk of various diseases, such as mood disorders, metabolic syndrome, cardiovascular disease, and some types of cancers, suggesting that the misalignment between environmental cycles and endogenous circadian clocks exacerbates systemic pathological consequences 3-8. However, the pathophysiological mechanisms underlying the deleterious effects of long-term circadian misalignment in health and healthspan remain unclear. Recent studies have investigated the perturbation of circadian systems by environmental and/or genetic manipulation in animal models 9,10. For example, Davidson et al. reported that an experimental model of environmental perturbation induced by the scheduled shifts of light-dark cycles-called chronic jet-lag (CJL)-for 8 weeks using aged mice (27-31 months old) showed the mortality rate to be higher in the phase advance condition (6-hour phase advance every 7 days) than in the phase delay (6-hour phase delay every 7 days) condition or control LD condition 9. These studies principally investigated the acute or subacute (for up to a few months) effects of circadian misalignment; it thus remains uncertain how long-term perturbation of environmental light-dark cycle induces physiological transformation and pathological consequences.

The circadian clock regulates behavioural and physiological processes in a 24-h cycle. The nuclear receptors REV-ERBα and REV-ERBβ are involved in the cell-autonomous circadian transcriptional/translational feedback loops as transcriptional repressors. A number of studies have also demonstrated a pivotal role of REV-ERBs in regulation of metabolic, neuronal, and inflammatory functions including bile acid metabolism, lipid metabolism, and production of inflammatory cytokines. Given the multifunctional role of REV-ERBs, it is important to elucidate the mechanism through which REV-ERBs exert their functions. To this end, we established a Rev-erbα / Rev-erbβ double-knockout mouse embryonic stem (ES) cell model and analyzed the circadian clock and clock-controlled output gene expressions. A comprehensive mRNA-seq analysis revealed that the double knockout of both Rev-erbα and Rev-erbβ does not abrogate expression rhythms of E-box-regulated core clock genes but drastically changes a diverse set of other rhythmically-expressed output genes. Of note, REV-ERBα/ β deficiency does not compromise circadian expression rhythms of PER2, while REV-ERB target genes, Bmal1 and Npas2 , are significantly upregulated. This study highlight the relevance of REV-ERBs as pivotal output mediators of the mammalian circadian clock.

Circadian clocks in mammals function in most organs and tissues throughout the body. Various renal functions such as the glomerular filtration and excretion of electrolytes exhibit circadian rhythms. Although it has been reported that the expression of the clock genes composing molecular oscillators show apparent daily rhythms in rodent kidneys, functional variations of regional clocks are not yet fully understood. In this study, using macroscopic bioluminescence imaging method of the PER2::Luciferase knock-in mouse kidney, we reveal that strong and robust circadian clock oscillation is observed in the medulla. In addition, the osmotic pressure in the inner medulla shows apparent daily fluctuation, but not in the cortex. Quantitative-PCR analysis of the genes contributing to the generation of high osmotic pressure or the water re-absorption in the inner medulla, such as vasopressin receptors (V1aR, V2R), urea transporter (UT-A2) and water channel (Aqp2) show diurnal variations as well as clock genes. Deficiency of an essential clock gene Bmal1 impairs day-night variations of osmotic pressure gradient in the inner medulla, suggesting that circadian clocks in the medulla part of the kidney may regulate the circadian rhythm of cortico-medullary osmotic pressure gradient, and may contribute physiological day-night rhythm of urination.