Photochemistry and Photobiology in Light and Life Research (OptoBioTechnology Research Center, Nagoya Institute of Technology) Kandori, Hideki

Biological systems utilize light as the source of signal and energy, as seen in our vision and plants’ photosynthesis, respectively. However, light is also harmful, because UV light damages DNA. Interestingly, biological system has an enzyme to repair the damaged DNA using light. Photochemical reactions of chromophore molecules in photoreceptive proteins initiate protein structural changes for various functions, whose mechanisms are of our particular interest.

Light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful,

sensitive and informative method to study structural changes of the chromophore, peptide backbone, side chains and protein-bound water molecules in photoreceptive proteins [1].

Using light-induced difference FTIR spectroscopy, we have studied various rhodopsins,

which function as visual and bacterial light sensors, light-driven ion pumps, or light-gated ion channels [2]. Such multiple functions are initiated by common photochemistry, cis-trans isomerization of retinal, and light-induced difference FTIR spectroscopy provided structural elements for each function. A recent topic is the role of protein-bound water molecule. Our comprehensive analysis revealed that a strongly-hydrogen-bonded water molecule is the functional determinant of proton pump, which explained asymmetric functional conversion of light-driven archaeal proton and chloride pumps [3, 4]. Another topic is a light-driven sodium pumping rhodopsin, which is of a new functional class in microbial rhodopsins and a potential tool in optogenetics [5, 6].

We also study flavin binding photoreceptors such as LOV domain, BLUF domain, and

photolyase (PHR) and cryptochrome (CRY), whose photochemistry differs with each other, unlike rhodopsins [7]. A recent topic is the repair mechanism of CPD and (6-4) PHR and their functional conversion by mutation.

In my talk, I will present recent understanding on photochemistry and photobiology of

these photoreceptive proteins. Protein structural changes of microbial rhodopsins and PHR/CRY family proteins will be presented, including our trial of functional conversion of these proteins.

1L01

[1] Y. Furutani, H. Kandori, Biochim. Biophys. Acta 2014, 1837, 598. [2] O. P. Ernst, D. T. Lodowski, M. Elstner, P. Hegemann, L. S. Brown, H. Kandori, Chem. Rev. 2014, 114, 126. [3] J. Sasaki, L. S. Brown, Y.-S. Chon, H. Kandori, A. Maeda, R. Needleman, J. K. Lanyi, Science 1995 269, 73. [4] K. Muroda, K. Nakashima, M. Shibata, M. Demura, H. Kandori, Biochemistry 2012, 51, 4677. [5] K. Inoue, H. Ono, R. Abe-Yoshizumi, S. Yoshizawa, H. Ito, K. Kogure, H. Kandori, Nat. Commun. 2013, 4, 1678. [6] H. E. Kato, K. Inoue, R. Abe-Yoshizumi, Y. Kato, H. Ono, M. Konno, T. Ishizuka, M. R. Hoque, S. Hososhima, H. Kunimoto, J. Ito, S. Yoshizawa, K. Yamashita, M. Takemoto, T. Nishizawa, R. Taniguchi, K. Kogure, A. D. Maturana, Y. Iino, H. Yawo, R. Ishitani, H. Kandori, O. Nureki, Nature 2015, 521, 48. [7] D. Yamada, H. Kandori, Methods Mol. Biol. 2014, 1148, 361.

Probing diversity and possibility of microbial retinal proteins (Okayama Univ.) SUDO, Yuki

[Introduction] Retinal proteins are photoreactive biological molecules with vitamin-A aldehyde retinal as a chromophore surrounded by seven transmembrane alpha-helices (Figure 1). They are roughly divided into two types, type-1 for microbes and type-2 for animals [1]. So far (~1999), the type-1 retinal proteins have only been described in halophilic archaea. After 1999, environmental genomics have revealed that they are broadly distributed among bacteria and eukarya. At present, it is well-known that they are widely distributed through all three biological kingdoms, eukarya, bacteria and archaea, indicating the biological significance of the retinal proteins [1]. Visible light absorption triggers trans-cis photoisomerization of the retinal and it induces the structural changes of the protein moiety, resulting in a variety of biological functions such as vision, ion transportation and photosensing. In addition to its biological aspect, retinal proteins have become a focus of interest in part because of application for optogenetics. On the basis of our results and other findings, we would like to highlight the recent progress in the structural and functional studies on type-1 retinal proteins. [Experimental] The genes encoding microbial retinal proteins are chemically synthesized with codon optimization or genetically cloned from the native cells. Several types of the cells including E. coli, H. salinarum, C. elegans and P. pastoris were employed for the protein expression. The preparation of crude membranes and the purification of proteins were performed using essentially a previously described method [2, 3]. Briefly, the cell membranes were solubilized by detergent such as n-dodecyl-β-D-maltoside and purified with a Ni2+ affinity column. When necessary, the samples were further purified with an anion exchange column as described previously [4]. The purified sample was concentrated and exchanged using an Amicon Ultra filter, against buffer solution.

Figure. 1. Protein structure of type-1 retinal protein and

light-induced isomerization of the chromophore retinal.

1L02

[Results and Discussion] Our retinal protein research is summarized in Figure 2. The continual discovery of microbial type-1 retinal proteins becomes a focus of interest in part because of their importance to the general understanding of light-energy conversion in nature. We found several novel retinal proteins and extensively characterized them using a variety of methods including biochemical techniques, vibrational spectroscopy, NMR spectroscopy and X-ray crystallography [5, 6]. The detailed structural and functional analysis allowed us to make a functional conversion [7] and to produce optical tools for controlling biological activities in living animals [8, 9]. [1] Y. Sudo, CRC Handobook of Organic Photochemistry and Photobiology, the 3rd edition,

CRC Press, Boca Raton, 2012, 1173. [2] Y. Sudo, Y. Furutani, A. Wada, M. Ito, N. Kamo, H. Kandori*, J. Am. Chem. Soc. 2005,

127, 16036. [3] Y. Sudo, J. L. Spudich*, Proc. Natl. Acad. Sci. USA 2006, 103, 16129. [4] Y. Sudo*, Y. Yuasa, J. Shibata, D. Suzuki, M. Homma, J. Biol. Chem. 2011, 286, 11328. [5] Y. Sudo*, K. Ihara, S. Kobayashi, D. Suzuki, H. Irieda, T. Kikukawa, H. Kandori, M.

Homma, J. Biol. Chem. 2011, 286, 25967. [6] T. Tsukamoto, K. Inoue, H. Kandori, Y. Sudo*, J. Biol. Chem. 2013, 288, 21581. [7] K. Inoue, T. Tsukamoto, K. Shimono, Y. Suzuki, S. Miyauchi, S. Hayashi, H. Kandori, Y.

Sudo*, J. Am. Chem. Soc. 2015, 137, 3291. [8] Y. Sudo*, A. Okazaki, H. Ono, J. Yagasaki, S. Sugo, M. Kamiya, L. Reissig, K. Inoue, K.

Ihara, H. Kandori, S. Takagi, S. Hayashi, J. Biol. Chem. 2013, 288, 20624. [9] H.E. Kato, M. Kamiya, S. Sugo, J. Ito, R. Taniguchi, A. Orito, K. Hirata, A. Inutsuka, A.

Yamanaka, A.D. Matsurana, Y. Sudo, S. Hayashi*, O. Nureki*, Nat. Commun. 2015, 6, 7177.

Figure. 2. Schematic diagram of our research.

Introduction of animal rhodopsins and their optogenetic

potentials

(Osaka City Univ.) KOYANAGI, Mitsumasa

[Introduction] In animals, the rhodopsin-like photopigments (here after called opsin-based

pigments) act as photoreceptors for vision and other non-visual functions such as circadian

photoentrainment and pupil response. Most animal opsin-based pigments are typical G

protein-coupled receptors (GPCRs) and consist of a protein moiety, opsin, and 11-cis retinal

as a chromophore. Upon light absorption, the chromophore in the opsin-based pigment

undergoes 11-cis to all-trans isomerization to form the photoproduct of the opsin-based

pigment (Fig.1). The photoproduct activates a G protein-mediated signal transduction cascade,

leading to alteration of the conditions within photoreceptor cells and generation of a cellular

response, generally an electrical response. Thousands of opsins have been identified and they

are roughly divided into eight groups. Basically, members belonging to different groups

activate different types of G-proteins such as transducin, Gi, Go, Gs and Gq, and visual opsin

groups contain different color-sensitive opsins.

Fig.1 Photochemical properties of bleaching and bistable pigments

[Experimental] We have tried to express various kinds of non-conventional visual opsin-

based pigments such as non-visual opsin-based pigments and invertebrate visual opsin-based

pigments in cultured cells and conducted spectroscopic analyses of recombinant opsin-based

pigments as well as measurements of light-induced cellular responses of opsin-expressing

cultured cells, in order to uncover and compare their molecular properties [1-6].

1L03

[Results and Discussion] Our analyses revealed that most opsin-based pigments excluding

vertebrate visual-opsin based pigment (rhodopsin and cone visual pigments) are a bleach-

resistant or bistable pigment, that is, the light-activated state is stable and, in many cases,

reverts to the original dark state by subsequent light irradiation (Fig. 1). In addition, we

recently found that a member of Opn3 group binds to 13-cis retinal, a ubiquitously present

retinal isomer to form a bistable pigment, suggesting that the Opn3 homologue potentially

serves as a light-sensor in non-photoreceptive tissues [6]. In fact, we demonstrated that the

introduction of Opn3 homologues rendered cultured mammalian kidney cells photosensitive

without the addition of 11-cis retinal to the culture medium, even after continuous light

exposure, which is in contrast to the observation that the introduction of a bleaching pigment

bovine rhodopsin did not. Here we discuss the optogenetic potentials of varied bistable

pigments including Opn3 homologues as well as engineered bistable pigments from

viewpoints of color sensitivity and selective activation of different G proteins.

[1] M. Koyanagi, E. Kawano et al., Proc. Natl. Acad. Sci. USA 2004, 101, 6687-6691

[2] M. Koyanagi et al., Curr. Biol. 2005, 15, 1065-1069

[3] M. Koyanagi et al., Proc. Natl. Acad. Sci. USA 2008, 105, 15576-15580

[4] A. Terakita et al., J. Neurochem. 2008, 105, 883-890

[5] T. Nagata et al., Science 2012, 335, 469-471

[6] M. Koyanagi et al., Proc. Natl. Acad. Sci. USA 2013, 110, 4998-5003

Subcellular optogenetics for controlling cell behavior (Washington Univ School of Medicine) Gautam, N

[Introduction] We have examined the possibility of using light sensitive proteins to modulate or perturb signaling at the subcellular level, optically direct an important cell behavior, cell migration and quantitatively monitor the molecular and cellular response with the aim of understanding how spatiotemporally dynamic signaling activities govern cell migration [1]. [Experimental] We used three different types of optogenetic strategies in immune cells and imaged the dynamic molecular and cellular responses that result from localized optical activation. The first strategy was based on GPCR opsins that activate GPCR pathways in their entirety [2,3]. The second was based on the cryptochrome domain, CRY2 and facilitates deactivation of heterotrimeric G protein alpha subunit or sequestration of the betagamma complex [4]. The third was based on an LOV domain dependent translocation scheme that allows selective activation of small G proteins, Cdc42, Rac or RhoA. A common characteristic of each of these experimental strategies was that they allowed modulation of the specific signaling protein activity asymmetrically across a single cell. [Results and Discussion] The results of these experiments showed that the different components of cell migration -- initiation, persistence and directional sensitivity could be directed optically. The differential responses to activation by different optogenetic approaches provided insights about the molecular mechanisms underlying migration. For example, differential responses to the CRY2 G alpha deactivation and betagamma sequestration suggest an unexpected role for the alpha subunit in cell migration. Surprising findings from the localized activation of Cdc42 and RhoA suggest unanticipated major roles for these proteins in cell migration. The development of a library of fluorescent protein tools has revolutionized the ability to view subcellular signaling activity in living cells. A library of optogenetic tools that control this dynamic subcellular signaling activity in living cells are proving to be transformative in enabling experimental control over important cellular behaviors and identifying the underlying molecular mechanisms. [1] W.K.A. Karunarathne, P.R. O'Neill, N. Gautam, J. Cell Sci. 2015, 128, 15. [2] W.A.K. Karunarathne, L. Giri, V. Kalyanaraman, N. Gautam, Proc. Natl. Acad. Sci. USA. 2013, 110, E1565. [3] W.A.K. Karunarathne, L. Giri, A.K. Patel, K.V. Venkatesh, N. Gautam, Proc. Natl. Acad. Sci. USA. 2013, 110:E1575. [4] P.R. O'Neill, N. Gautam, Mol. Biol. Cell. 2014, 25, 2305.

1L04

Optogenetic patterning of neural activity (Tohoku Univ.) YAWO, Hiromu; YOKOYAMA, Yukinobu; ABE, Kenta,

LIU, Yueren

[Introduction] All knowledge about the world is perceived through our sensory systems

which consist of peripheral sensory organs, sensory nerves and central nervous system (CNS).

In principle a sensation is classified according to its modality, a kind of energy inducing

physiological transduction in a specific group of sensory organs. For example, in the

somatosensory systems, each mode of touch-pressure, temperature or pain is sensed by

independent sensory endings of primary afferent neurons and conducted to the specific

cortical locus as nerve impulses, but integrated thereafter as a whole. The spatiotemporal

patterns of touch sense generate the complex tactile perception such as form, movement, size

and texture. How is the complex perception generated from simple information of points? Is it

formed by the experience? We are challenging these enigmas by applying optogenetic pattern

to each whisker of a rat under recording cortical responses.

[Experimental] We have previously generated several lines of transgenic rats which express

channelrhodopsin-2 (ChR2), one of algal photoreceptive molecules1,2, under regulation of

thy1.2 promoter3. The transgene expression was variable from line to line, being dependent on

the integration sites in chromosomes and/or the number of inserted copies. In one of these

transgenic rat lines, W-TChR2V4, ChR2 was specifically expressed in a subpopulation of

mechanoreceptive neurons in the dorsal root ganglion (DRG) but not in the small-sized

neurons which are involved in nociception. Furthermore, ChR2 is also expressed in their

peripheral nerve endings such as those are innervating Merkel corpuscles and Meissner

corpuscles, which are involved in the touch sense. Indeed, this transgenic rat showed a

sensory-evoked behavior in response to blue flash light on their plantar skin as if it were

touched by something. However, it ignores red light that is not sensed by ChR2. It is thus

concluded that the rat has acquired an unusual sensory modality that it senses light at skin4.

We also identified the expression of ChR2 in the

peripheral endings of trigeminal

mechanoreceptive neurons which innervate

whisker follicles.

Sixteen whisker follicles of W-TChR2V4

rat were one-by-one connected to blue LEDs via

optical fibers and each LED was lighten up with

a given temporal pattern (Fig. 1). The cortical Fig. 1 Fiber-coupled LED system for

whisker photostimulation

1L05

responses to a certain pattern were then evaluated by the electrophysiological methods,

functional magnetic resonance imaging (fMRI) and behavioral responses.

[Results and Discussion] We found that the blue light irradiation of whisker follicles evoked

enhanced unit activities as well as a local field potential in the barrel field of contralateral

somatosensory cortex whereas the red light did not5. The blue light irradiation also induced

blood oxygenation level-dependent (BOLD) responses in the barrel field of contralateral

somatosensory cortex. A W-TChR2V4 rat was irradiated blue light on each whisker with a

certain pattern conditioned with a reward. The Go task was designed so as the rat is allowed

to get a reward, when it licked the nozzle within 5 s after irradiation of one of whiskers. The

No-go task was designed so as the rat have to withhold licking at least 5 s to get a reward after

irradiation of another whisker. The rat learned to discriminate these optogenetic whisker

patterns successively with sessions and even with days. It is suggested that the optogenetic

whisker stimulation could activate the whisker-barrel cortical pathway of mechanoreceptive

signaling.

The light-evoked somatosensory would facilitate the study how the complex tactile

perception such as form, movement, size and texture is generated6. The various and

reproducible patterned tactile stimulations could be easily made by the patterned illuminations

on the whisker array without using any mechanical instruments. Since our rat system does not

express ChR2 in the nociceptive pathway, it enables one to do in vivo experiments without

ethical problems. It would particularly beneficial for the researches using fMRI because the

illumination system would not influence the magnetic fields. The light-evoked somatosensory

perception should facilitate study of how the complex tactile sense emerges in the brain.

[1] Nagel G, et al. (2003) Channelrhodopsin-2, a directly light-gated cation-selective

membrane channel. Proc. Natl. Acad. Sci. USA 100:13940–13945.

[2] Ishizuka T, et al. (2006) Kinetic evaluation of photosensitivity in genetically engineered

neurons expressing green algae light-gated channels. Neurosci. Res. 54:85-94.

[3] Tomita H, et al. (2009) Visual properties of transgenic rats harboring the

Channelrhodopsin-2 gene regulated by the Thy-1.2 promoter. PLoS One 4: e7679.

[4] Ji Z-G., et al. (2012) Light-evoked somatosensory perception of transgenic rats which

express channelrhodopsin-2 in dorsal root ganglion cells. PLoS ONE 7:e32699.

[5] Honjoh T, et al. (2014) Optogenetic patterning of whisker-barrel cortical system in

transgenic rat expressing channelrhodopsin-2. PLoS ONE. 9 (4): e93706.

[6] Ji Z-G., et al. (2015) Strategies to probe mechanoreception: from mechanical to

optogenetic approaches. “Optogenetics: Light-Sensing Proteins and Their Applications”,

Yawo H., Kandori H. and Koizumi A. eds, Springer, Tokyo, pp.305-314.

Optogenetical manipulation of neural activity and behavior control (Research Institute of Environmental Medicine, Nagoya Univ.) YAMANAKA, Akihiro; TSUNEMATSU, Tomomi; INUTSUKA, Ayumu; YAMASHITA, Akira

[Introduction] We have examined the regulatory mechanism of instinctive behaviors. Instinctive behaviors, such as feeding and drinking behaviors, sleep/wakefulness and sexual behavior, are important behaviors to survive or to keep species. These behaviors are regulated by the neurons in the hypothalamus. Some neurons in the hypothalamus contain neuropeptides and release them as a neurotransmitter. Recent studies revealed that these neuropeptides have a crucial role in the regulation of instinctive behaviors. In spite of its physiological and biological importance, little is known about the regulatory mechanism of instinctive behaviors since these behaviors are only exhibited in the whole animal. Recently developed technique, “Optogenetics” is a powerful technique to study regulatory mechanism of these behaviors in vivo. [Experimental] Optogenetics enables manipulate the activity of neurons by expressing light–gated channel or ion pump in the specific type of neurons with high temporal accuracy. Optogenetics allow us to study neural regulatory mechanism of instinctive behaviors using a whole animal. To apply optogenetics in various type of cell in the brain, we generated new transgenic mice using tetracycline gene expression system. These mice enable express channelrhodopsin2 (ChR2) or Archaerhodopsin-3 (ArchT) by bred with various lines of tTA mice in which the specific type of neurons express tTA. We generated tTA mice lines which specifically express tTA in the peptide containing neurons in the hypothalamus using its promoter, such as prepro-orexin promoter or melanin concentrating hormone promoter. And we applied optogenetics in these peptide-containing neurons to reveal regulatory mechanism of sleep/wakefulness. [Results and Discussion] Activation of orexin neurons using optogenetics or pharmacogenetics increased time in wakefulness. On the other hand, inhibition of orexin neurons increased time in non-REM sleep in mice. These data indicated that orexin neurons are involved in the state switching from wakefulness to non-REM sleep. Recent studies report that melanin concentrating hormone (MCH) containing neurons are involved in state switching from non-REM sleep to REM sleep. To study the role of MCH neurons in sleep/wakefulness, we applied optogenetics to MCH neurons. Activation of MCH neurons using optogenetics increased time spent in REM sleep. REM sleep was

1L06

induced by MCH neurons was activated when mice were non-REM sleep. Interestingly, however, inhibition of MCH neurons using optogenetics did not decreased time spent in REM sleep suggesting that MCH neurons are not a center of REM sleep but a modulator of REM sleep. These results suggest that peptide containing neurons (orexin neurons and MCH neurons) in the hypothalamus play important role in the regulation of state change among wakefulness, non-REM sleep and REM sleep. [1] Tsunematsu T, Kilduff TS, Boyden ES, Takahashi S, Tominaga M, Yamanaka A, Acute

optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice. J. Neurosci. 2011, 31, 10529-10539.

[2] Tsunematsu T, Tabuchi S, Tanaka KF, Boyden ES, Tominaga M, Yamanaka A, Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice. Behav. Brain Res. 2013, 255, 64-74.

[3] Tsunematsu T, Ueno T, Tabuchi S, Inutsuka A, Tanaka KF, Hasuwa H, Kilduff TS, Terao A, Yamanaka A, Optogenetic manipulation of activity and temporally controlled cell-specific ablation reveal a role for MCH neurons in sleep/wake regulation. J Neurosci, 2014, 34, 6896-6909.

Tuning mechanisms underlying photoreceptor spectral sensitivity (Sokendai) ARIKAWA, Kentaro

[Introduction] Butterflies have sophisticated color vision. Their compound eyes consist of small units called ommatidia, each containing 9 photoreceptor cells. The photoreceptor cells are quite variable in spectral sensitivity, which serve the physiological basis of color vision. In the course of studying the mechanisms underlying color vision, we have found at least three basics for spectral tuning of photoreceptors. The mechanisms are 1) the property of visual pigment molecule per se, 2) expression pattern of visual pigment opsins and 3) spectral filtering by photostable pigments [1]. [Experimental] The spectral properties of their photoreceptors were studied by combining single cell electrophysiology, light and electron microscopy, identification and localization of visual pigment opsin molecules, optical physiology and computer simulation. [Results and Discussion] The eyes of the Japanese yellow swallowtail butterfly, Papilio xuthus, are furnished with six spectral classes of photoreceptors, which are of the ultraviolet (UV), violet (V), blue (B), green (G), red (R) and broad-band (BB) classes. On the other hand, their eyes express one UV (PxUV), one B (PxB) and three long wavelength-absorbing (PxL1-3) visual opsins: there is no one-to-one relationship between the numbers of spectral receptors and opsins. UV, B, G and R receptors express the PxUV, PxB, PxL2 and PxL3, respectively. Some G receptors coexpress PxL1 with L2. The PxUV is also expressed in V receptors, which are embedded in the ommatidia containing 3OH-retinol. The retinol acts as an UV-absorbing filter for PxUV producing the aberrantly narrow V spectral sensitivity. BB receptors coexpress PxL2 and L3, which together form the broad sensitivity stretching from 420 to 650 nm. The Papilio eyes are not sexually dimorphic, but it is rather commonly found in the eyes of white butterflies (Pieridae). Figure 1 shows an example of the Eastern pale-clouded yellow butterfly, Colias erate. Each ommatidium contains four clusters of reddish pigment in the center. According to the arrangement of the clusters, the ommatidia can be divided into three types. The color of the pigment is identical in all three types in males (A), but it is orange in female type II (B). We identified the spectral sensitivities of all photoreceptors in three types of ommatidia both in males (C-E) and females (F-H). The variability of receptors peaking at wavelengths longer than 600 nm can be well explained by the reddish pigments. [1] K. Arikawa, D.G. Stavenga. In Evolution of visual and non-visual pigments. (Hunt et al, Eds), Springer 2014, 137-162. (ISBN 978-1-4614-4354-4)

1L07

Figure 1 Photoreceptors of Colias erate. Sections of the eyes show that the ommatidia contain reddish pigments, whose color is orange only in female type II ommatidia (A, B). Electrophysiologically-determined spectral sensitivities of photoreceptors in type I, II and III ommatidia in males (C-E) and in females (F-H). The table summarizes the photoreceptor arrangement. R1-R8: photoreceptor number in the ommatidia. CeUV, CeV1, CeV2, CeB, CeL: visual pigment opsins.

Light signaling molecule in biological clocks and its role in clock oscillation (Univ. Tokyo) FUKADA, Yoshitaka

[Introduction] The intrinsic periods of circadian clocks are generally diverged from 24 hours, but the clocks can be synchronized (entrained) to 24-hr period of the ambient signals such as day-night cycle. For the entrainment, the circadian clock is phase-delayed or -advanced by light given at early or late night, respectively. The time-of-day-dependent phase-response to light is an important property common to all the circadian clock systems. Despite accumulation of molecular level studies on the animal circadian clockwork, the mechanisms underlying the stable circadian oscillation and its phase regulation are still poorly understood. Among clock tissues in vertebrates, the chicken pineal gland is unique in that it retains intrinsic phototransduction pathways for entrainment of the intracellular clockwork. Therefore the chicken pinealocyte provides a prominent platform for studies on the light-entrainment mechanism. Our challenge to comprehensive analysis of the pineal light-inducible genes revealed phase-dependent activation of transcription factors HSF1, HSF2, XBP1 and E4BP4, a series of regulators essential for clock gene expression [1,2]. In contrast, the mammalian central clock, the hypothalamic suprachasmatic nucleus (SCN), is intrinsically insentitive to light, but it receives light-signal captured by the retina through glutamatergic innervation. The cell-autonomous circadian oscillation in the SCN is generated by a transcription/translation-based autoregulatory feedback mechanism. Basic helix-loop-helix transcription factors, CLOCK and BMAL1, form a heterodimer to activate transcription of Period (Per) and Cryptochrome (Cry) genes through their E-box enhancer elements. Translated PER and CRY proteins then translocate into the nucleus, where they interfere with CLOCK/BMAL1-dependent transcription and thereby repress their own expression. The mammalian SCN clockwork is characterized by stably sustainable oscillation, which is achieved by a network of posttranslational regulations of clock proteins: A pair of central clock proteins, CRY1 and CRY2, are phosphorylated by a series of protein kinases [3] and ubiquitinated by FBXL3/FBXL21, two closely related ubiquitin E3 ligases. These modifications steer CRYs to the degradation and stabilization, respectively, providing antagonizing effects that are important for formation of the stable oscillation of the SCN neurons [4]. [Results and Discussion] Daily behavioral rhythms of mice persist even in constant darkness with a stable activity time due to coupling between two oscillators within the SCN; the evening and morning oscillators that respectively determine the timing of the activity onset and offset in the nocturnal animals (“a two oscillators model”). Intriguingly, individual neurons in a cultured SCN slice exhibit robust circadian fluctuation of intracellular Ca2+

1L08

concentration, while chelating of intracellular Ca2+ abrogates rhythmic expression of clock genes. These studies led us to speculate that a Ca2+-dependent feedback system regulates the transcriptional rhythms and raise a fundamental question as to how the Ca2+ signaling is integrated into the transcriptional feedback mechanism. On the other hand, when the SCN neurons receive neurotransmitter Glu as the light signal from the terminals of retino-hypothalamic tract, Ca2+ influx and subsequent activation of Ca2+/calmodulin-dependent protein kinase, CaMKII, mediate the intracellular signaling triggered by GluR activation. In addition to such a role in light-input process, we found that CaMKII activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKII-alpha gene weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms. The mutant mice sometimes lost the behavioral rhythmicity in constant darkness. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. These results demonstrate the eminent roles of CaMKII as a component of cell autonomous clockwork and as a synchronizer for the mutual coupling among SCN neurons [5].

Figure 1. A model for the role of CaMKII in the mouse circadian clock. [1] M. Doi, T. Okano, I. Yujnovsky, P. Sassone-Corsi & Y. Fukada, Curr. Biol. 2004, 14, 975 [2] M. Hatori, T. Hirota, M. Iitsuka, N. Kurabayashi, S. Haraguchi, K. Kokame, R. Sato, A.

Nakai, T. Miyata, K. Tsutsui & Y. Fukada, Proc. Natl. Acad. Sci. USA., 2011, 108, 4864 [3] A. Hirano, N. Kurabayashi, T. Nakagawa, G. Shioi, T. Todo, T. Hirota & Y. Fukada,

Mol. Cell. Biol., 2014, 34, 4464 [4] A. Hirano, K. Yumimoto, R. Tsunematsu, M. Matsumoto, M. Oyama, H. Kozuka-Hata, T.

Nakagawa, D. Lanjakornsiripan, K. I. Nakayama & Y. Fukada, Cell, 2013, 152, 1106 [5] N. Kon, T. Yoshikawa, S. Honma, Y. Yamagata, H. Yoshitane, K. Shimizu, Y. Sugiyama,

C. Hara, I. Kameshita, K. Honma & Y. Fukada, Genes Dev., 2014, 28, 1101