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In der Optogenetik, einer Kombination aus Optik und Genetik, werden Zellen durch genetische Manipulation lichtempfindlich, sodass Zellfunktionen gezielt durch Licht gesteuert, Krankheiten analysiert und neue Ansätze für Heilmethoden entwickelt werden können. Ein optogenetisches System besteht meist aus zwei Transkriptionsfaktoren, die unter Lichtaktivierung eines Chromophors eine Einheit bilden, der den Promotor der Zielsequenz aktiviert und die Proteinsynthese startet. Ziel der vorliegenden Arbeit war die Entwicklung einer lichtinduzierten Proteinsynthese in optogenetisch aktivierbaren humanen Zellen für therapeutische Applikationen. Es könnten z. B. patienteneigene mesenchymale Stammzellen (hMSCs) während der Implantation auf dem CI (Cochleaimplantat) platziert werden, die auf Lichtinduktion hin BDNF (brain-derived neurotropic factor) synthetisieren. BDNF hat einen positiven Effekt auf Spiralganglionzellen, die wiederrum die auditorische Vermittlung von CIs verbessern. Durch die gute zeitliche und räumliche Auflösung optogenetischer Systeme wird umliegendes Gewebe geschont und ungewollte Nebeneffekte minimiert. Als Modellzellen wurden Säugertierzellen verwendet. Eine Etablierung des optogenetischen PhyB (phytochrome B)-Systems in CHO-K1 (Chinese Hamster Ovary) Zellen mit EGFP (green fluorescent protein) Marker führte nach Optimierung zu einer 5-mal höheren Proteinexpression im Vergleich zur Leakage, während beim CRY2 (Cryptochrome 2)-System in HEK293 (human embryonic kidney) Zellen mit Luciferase (Luc) Marker eine um den Faktor 26 gesteigerte Proteinexpression erzielt wurde. Eine Analyse der Genexpression mittels qPCR (quantitative Polymerase-kettenreaktion) bestätigte diese Ergebnisse. Beide Systeme konnten sowohl mittels LED-Induktion (light emitting diode) wie auch durch Laser-Induktion aktiviert werden und das Protein BDNF optogenetisch synthetisiert werden, wobei nur das CRY2-System eine therapeutisch relevante Konzentration erzeugte. Für eine Übertragung die, In optogenetics, a combination of optics and genetics, cells become light-sensitive through genetic manipulation, so cell functions can be specifically controlled by light, diseases can be analyzed and new approaches for cures can be developed. An optogenetic system consists usually of two transcription factors that assemble into a unit under light activation of a chromophore, which activates the promoter of the target sequence and starts protein synthesis. The aim of the present work was the development of a light-induced protein synthesis in optogenetically activatable human cells for therapeutic applications. For example patient-derived mesenchymal stem cells (hMSCs) could be placed on the CI (cochlear implant) during implantation and synthesize BDNF (brain-derived neurotropic factor) upon light induction. BDNF has a positive effect on spiral ganglion cells which in turn enhance auditory mediation of CIs. The good temporal and spatial resolution of optogenetic systems spares surrounding tissue and minimizes unwanted side effects. Mammalian cells were used as model cells. Establishment of the optogenetic PhyB (phytochrome B) system in CHO-K1 (Chinese hamster ovary) cells with EGFP (green fluorescent protein) marker resulted in 5-fold higher protein expression after optimization compared to leakage while a 26-fold increase in protein expression, was obtained for the CRY2 (cryptochrome 2)-system in HEK293 (human embryonic kidney) cells with luciferase marker. Analysis of gene expression by qPCR (quantitative polymerase chain reaction) confirmed these results. Both systems could be activated by LED (light emitting diode) induction as well as by laser induction and synthesized the protein BDNF optogenetically whereas only the CRY2-system produced a therapeutically relevant concentration. Various transfection methods were tested to transfer these systems into hMSCs here isolated from adipose tissue. Chemical transfection (using DreamFect™ Gold) provided the best result

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo(RNAi) crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

Aims/hypothesis The association of single nucleotide polymorphisms (SNPs) proximal to CRY2 and MTNR1B with fasting glucose is well established. CRY1/2 and MTNR1B encode proteins that regulate circadian rhythmicity and influence energy metabolism. Here we tested whether season modified the relationship of these loci with blood glucose concentration. Methods SNPs rs8192440 (CRY1), rs11605924 (CRY2) and rs10830963 (MTNR1B) were genotyped in a prospective cohort study from northern Sweden (n = 16,499). The number of hours of daylight exposure during the year ranged from 4.5 to 22 h daily. Owing to the non-linear distribution of daylight throughout the year, season was dichotomised based on the vernal and autumnal equinoxes. Effect modification was assessed using linear regression models fitted with a SNP x season interaction term, marginal effect terms and putative confounding variables, with fasting or 2 h glucose concentrations as outcomes. Results The rs8192440 (CRY1) variant was only associated with fasting glucose among participants (n = 2,318) examined during the light season (beta = -0.04 mmol/l per A allele, 95% CI -0.08, -0.01, p = 0.02, p (interaction) = 0.01). In addition to the established association with fasting glucose, the rs11605924 (CRY2) and rs10830963 (MTNR1B) loci were associated with 2 h glucose concentrations (beta = 0.07 mmol/l per A allele, 95% CI 0.03, 0.12, p = 0.0008, n = 9,605, and beta = -0.11 mmol/l per G allele, 95% CI -0.15, -0.06, p < 0.0001, n = 9,517, respectively), but only in participants examined during the dark season (p (interaction) = 0.006 and 0.04, respectively). Repeated measures analyses including data collected 10 years after baseline (n = 3,500) confirmed the results for the CRY1 locus (p (interaction) = 0.01). Conclusions/interpretation In summary, these observations suggest a biologically plausible season-dependent association between SNPs at CRY1, CRY2 and MTNR1B and glucose homeostasis.

The phase-resetting properties of the circadian system in mice with a functional deletion in mCry1, mCry2, mPer1, or mPer2 were studied in 2 experiments. In experiment 1, mCry1-/- and mCry2-/- mice as well as mPer1Brdm1 and mPer2Brdm1 mutant mice were exposed to 15-min light pulses during the 1st cycle following entrainment, either early (external time [ExT] 20) or late (ExT 4) in the subjective night. In experiment 2, a full PRC was measured for all these strains by exposure to light pulses of the same duration and intensity in free-running conditions in constant darkness. Directly after entrainment (experiment 1), mPer1 Brdm1 animals did not show significant phase advances by a light pulse in the late subjective night (ExT 4), as in the study by Albrecht et al. In the same experiment, mPer2Brdm1 mice became arrhythmic too frequently to reliably measure their phase responses. Mice with a targeted gene disruption in mCry1 or mCry2 showed increased phase delays compared to wild type after exposure to a light pulse in the early subjective night (ExT 20). Otherwise, phase shifts were not significantly affected. In free run (experiment 2), all genotypes did show phase advances and phase delays. The mPer2 Brdm1 mutant PRC was above the mPer1Brdm1 mutant and wild-type PRC (i.e., less delayed and more advanced) at most circadian phases. The mPer1Brdm1 mutant PRC was not distinguishable from the wild-type PRC. The mCry2-/- mice showed much smaller phase delays than did mCry1-/- mice in the subjective evening (delay phase). In general, mPer2Brdm1 mutant mice were more accelerated by light compared to mPer1Brdm1 and wild-type control mice, whereas mCry1-/- mice were more delayed by light than were mCry2-/- mice.