Two-dimensional angle measurement with sub-arcsecond precision and MHz update rate using heterodyne interferometry with optical frequency comb.

Heterodyne interferometry is a powerful tool for achieving high precision and fast measurement. We developed an angle measurement system based on heterodyne interferometry by combining discrete equal-spacing longitudinal modes of optical frequency comb with an acousto-optic modulator. Using a self-designed grating-corner-cube sensor, this method can achieve a two-dimensional angle measurement with sub-arcsecond accuracy and megahertz (MHz) update rate. We experimentally demonstrate a precision of 0.073 arcsec under a 3 MHz update rate, and comparison residuals are kept within 0.063 arcsec over 300 arcsec when compared to a piezo stage. In the dynamic measurement of a 40 Hz frequency, the continuous sinusoidal motion of 0.05 arcsec can be clearly distinguished and reconstructed.

Femtosecond-laser-based full-field three-dimensional imaging with phase compensation.

Coherence scanning interferometer (CSI) enables 3D imaging with nanoscale precision. However, the efficiency of such a system is limited because of the restriction imposed by the acquisition system. Herein, we propose a phase compensation method that reduces the interferometric fringe period of femtosecond-laser-based CSI, resulting in larger sampling intervals. We realize this method by synchronizing the heterodyne frequency with the repetition frequency of the femtosecond laser. The experimental results show that our method can keep the root-mean-square axial error down to 2 nm at a high scanning speed of 6.44 µm per frame, which enables fast nanoscale profilometry over a wide area.

Absolute angular position measurement by dual-comb spectroscopy of an autocollimation diffracted beam.

The dual-comb technique is a powerful tool in industrial inspection and scientific research and is capable of realizing ultrahigh-resolution and fast broadband spectral measurements. We propose an absolute angular-position measurement method based on dual-comb spectroscopy. With a simple layout, the absolute angular position can be naturally determined through the traceable and wide-amplitude spectra of the autocollimation diffracted beams of the target grating. We experimentally demonstrate that a precision of 0.12 arcsec in the dynamic range of approximately 6660 arcsec, along with a 1 kHz repetition rate difference, is achieved. Compared with a commercial autocollimator, over 1000 arcsec, the comparison residuals are kept within ±0.3 arcsec.

Dynamic ellipsometry measurement based on a simplified phase-stable dual-comb system.

Spectroscopic ellipsometry is a powerful tool for characterizing thin film, polarization optics, semiconductors, and others. Conventional approaches are subject to restrictions of mechanical instability and measurement speed. The complex locking scheme of previous dual-comb spectroscopic ellipsometry belies its practicability. We present and demonstrate here dynamic spectroscopic ellipsometry based on a simplified phase-stable dual-comb system, which could realize the online dynamic measurement of optical properties of materials. A precision of 1.31 nm and a combined uncertainty of 13.80 nm (k = 2) in the thickness measurement of thin-film samples has been achieved. Moreover, the dynamic performance of the system is investigated under a high data acquisition rate (1 kHz) with a dynamic resolution of ellipsometric parameter better than 0.1 rad.

Fast measurement of the thickness and group refractive index distribution of solid plates by line-field dispersive interferometry.

Real-time measurement of the thickness and group refractive index is crucial for semiconductor devices. In this paper, we proposed a fast synchronous method for measuring the thickness and group refractive index distribution of solid plates based on line-field dispersive interferometry. The proposed method measured the line-field distribution in an illuminated region through a single step. A low-cost spectrometer calibration method using an eight-channel dense wavelength division multiplexer was developed for verification. The line-field distribution of a three-step silicon wafer was successfully measured within 3.3 ms. The combined uncertainties for the geometrical thickness and group refractive index were <50 nm and 4 × 10-4, respectively.

Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells.

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

Design of a multiple self-mixing interferometer for a fiber ring laser.

In this Letter, a novel multiple self-mixing interferometer for a fiber ring laser (FRL) is designed by introducing a circular feedback cavity. A system model is established based on an injection-seeded erbium-doped FRL proposed by Dragic. Owing to the reflection of the collimating lens, the multiplied fringes have different depths, which shows two Doppler frequencies in the spectrum of the self-mixing signal. A double-peak frequency identification algorithm is proposed to extract the Doppler frequency from the unique signal. This technique has the potential to improve the accuracy of fiber self-mixing measurement systems, particularly in Doppler velocimeters.

Micro-displacement reconstruction using a laser self-mixing grating interferometer with multiple-diffraction.

In this paper, we demonstrated an improved laser self-mixing grating interferometer (SMGI) with auto-collimation design which can avoid the disturbance from the light feedback of the zero-order diffraction beam. In order to obtain higher optical subdivision, SMGI with multiple-diffraction is implemented. Both theoretical analysis and experimental work show that the proposed system for displacement measurement can achieve high sensitivity and low measurement uncertainty. Using the proposed system, different forms of micro-displacement signals applied on the target (grating) have been reconstructed with accuracy of a few nanometers. The work presented in this paper provides a good way to achieve robust and high precision measurement with compact system configuration.

Peptide Lv augments L-type voltage-gated calcium channels through vascular endothelial growth factor receptor 2 (VEGFR2) signaling.

We previously identified peptide Lv, a novel bioactive peptide that enhances the activity of L-type voltage-gated calcium channels (L-VGCCs) in cone photoreceptors. In this study, we verified that peptide Lv was able to augment L-VGCC currents in cardiomyocytes, as well as promote proliferation of endothelial cells. We used a proteomics approach to determine the specific receptors and binding partners of peptide Lv and found that vascular endothelial growth factor receptor 2 (VEGFR2) interacted with peptide Lv. Peptide Lv treatment in embryonic cardiomyocytes stimulated tyrosine autophosphorylation of VEGFR2 and activated its downstream signaling. Peptide Lv activity was blocked by DMH4, a VEGFR2 specific blocker, but not by SCH202676, an allosteric inhibitor of G protein-coupled receptors, suggesting that the activity of peptide Lv was mediated through VEGFR2 signaling. Inhibition of VEGFR tyrosine kinase or its downstream signaling molecules abolished the augmentation of L-VGCCs elicited by peptide Lv in cardiomyocytes. In addition, peptide Lv promoted cell proliferation of cultured human endothelial cells. Calcium entry through L-VGCCs is essential for excitation-contraction coupling in cardiomyocytes. Since peptide Lv was able to augment L-VGCCs through activation of VEGF signaling in cardiomyocytes and promote proliferation of endothelial cells, peptide Lv may play an important role in regulating the cardiovascular system.

A new role for AMP-activated protein kinase in the circadian regulation of L-type voltage-gated calcium channels in late-stage embryonic retinal photoreceptors.

AMP-activated protein kinase (AMPK) is a cellular energy sensor, which is activated when the intracellular ATP production decreases. The activities of AMPK display circadian rhythms in various organs and tissues, indicating that AMPK is involved in the circadian regulation of cellular metabolism. In vertebrate retina, the circadian clocks regulate many aspects of retinal function and physiology, including light/dark adaption, but whether and how AMPK was involved in the retinal circadian rhythm was not known. We hypothesized that the activation of AMPK (measured as phosphorylated AMPK) in the retina was under circadian control, and AMPK might interact with other intracellular signaling molecules to regulate photoreceptor physiology. We combined ATP assays, western blots, immunostaining, patch-clamp recordings, and pharmacological treatments to decipher the role of AMPK in the circadian regulation of photoreceptor physiology. We found that the overall retinal ATP content displayed a diurnal rhythm that peaked at early night, which was nearly anti-phase to the diurnal and circadian rhythms of AMPK phosphorylation. AMPK was also involved in the circadian phase-dependent regulation of photoreceptor L-type voltage-gated calcium channels (L-VGCCs), the ion channel essential for sustained neurotransmitter release. The activation of AMPK dampened the L-VGCC currents at night with a corresponding decrease in protein expression of the L-VGCCα1 pore-forming subunit, while inhibition of AMPK increased the L-VGCC current during the day. AMPK appeared to be upstream of extracellular-signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Hence, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology. We found that in chicken embryonic retina, the activation of AMP-activated protein kinase (AMPK) is under circadian control and anti-phase to the retinal ATP rhythm. While ATP content is higher at night, phosphorylated AMPK (pAMPK) is higher during the day. AMPK appears to be upstream of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and mammalian target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Therefore, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology.

Circadian phase-dependent effect of nitric oxide on L-type voltage-gated calcium channels in avian cone photoreceptors.

Nitric oxide (NO) plays an important role in phase-shifting of circadian neuronal activities in the suprachiasmatic nucleus and circadian behavior activity rhythms. In the retina, NO production is increased in a light-dependent manner. While endogenous circadian oscillators in retinal photoreceptors regulate their physiological states, it is not clear whether NO also participates in the circadian regulation of photoreceptors. In this study, we demonstrate that NO is involved in the circadian phase-dependent regulation of L-type voltage-gated calcium channels (L-VGCCs). In chick cone photoreceptors, the L-VGCCα1 subunit expression and the maximal L-VGCC currents are higher at night, and both Ras-mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (Erk) and Ras-phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) are part of the circadian output pathways regulating L-VGCCs. The NO-cGMP-protein kinase G (PKG) pathway decreases L-VGCCα1 subunit expression and L-VGCC currents at night, but not during the day, and exogenous NO donor or cGMP decreases the phosphorylation of Erk and Akt at night. The protein expression of neural NO synthase (nNOS) is also under circadian control, with both nNOS and NO production being higher during the day. Taken together, NO/cGMP/PKG signaling is involved as part of the circadian output pathway to regulate L-VGCCs in cone photoreceptors. In cone photoreceptors, the protein expression of neural nitric oxide synthase (nNOS) and NO production are under circadian control. NO-cGMP-protein kinase G (PKG) signaling serves in the circadian output pathway to regulate the circadian rhythms of L-type voltage-gated calcium channels (L-VGCCs) in part through regulating the phosphorylation states of extracellular-signal-regulated kinase (Erk) and protein kinase B (Akt).

Rhythmic expression of microRNA-26a regulates the L-type voltage-gated calcium channel alpha1C subunit in chicken cone photoreceptors.

MicroRNAs (miRNAs) modulate gene expression by degrading or inhibiting translation of messenger RNAs (mRNAs). Here, we demonstrated that chicken microRNA-26a (gga-mir-26a) is a key posttranscriptional regulator of photoreceptor L-type voltage-gated calcium channel alpha1C subunit (L-VGCCalpha1C) expression, and its own expression has a diurnal rhythm, thereby explaining the rhythmic nature of L-VGCCalpha1Cs. Circadian oscillators in retinal photoreceptors provide a mechanism that allows photoreceptors to anticipate daily illumination changes. In photoreceptors, L-VGCC activities are under circadian control, which are higher at night and lower during the day. Interestingly, the mRNA level of VGCCalpha1D oscillates, but those for VGCCalpha1C do not. However, the protein expression of both VGCCalpha1C and alpha1D are higher at night in cone photoreceptors. The underlying mechanism regulating L-VGCCalpha1C protein expression was not clear until now. In vitro targeting reporter assays verified that gga-mir-26a specifically targeted the L-VGCCalpha1C 3'-untranslated region, and gga-mir-26a expression in the retina peaked during the day. After transfection with gga-mir-26a, L-VGCCalpha1C protein expression and L-VGCC current density decreased. Therefore, the rhythmic expression of gga-mir-26a regulated the protein expression of the L-VGCCalpha1C subunit. Additionally, both CLOCK (circadian locomoter output cycles kaput) and CREB (cAMP-response element-binding protein-1) activated gga-mir-26a expression in vitro. This result implies that gga-mir-26a might be a downstream target of circadian oscillators. Our work has uncovered new functional roles for miRNAs in the regulation of circadian rhythms in cone photoreceptors. Circadian regulated miRNAs could serve as the link between the core oscillator and output signaling that further govern biological functions.

Circadian regulation of ion channels and their functions.

Ion channels are the gatekeepers to neuronal excitability. Retinal neurons of vertebrates and invertebrates, neurons of the suprachiasmatic nucleus (SCN) of vertebrates, and pinealocytes of non-mammalian vertebrates display daily rhythms in their activities. The interlocking transcription-translation feedback loops with specific post-translational modulations within individual cells form the molecular clock, the basic mechanism that maintains the autonomic approximately 24-h rhythm. The molecular clock regulates downstream output signaling pathways that further modulate activities of various ion channels. Ultimately, it is the circadian regulation of ion channel properties that govern excitability and behavior output of these neurons. In this review, we focus on the recent development of research in circadian neurobiology mainly from 1980 forward. We will emphasize the circadian regulation of various ion channels, including cGMP-gated cation channels, various voltage-gated calcium and potassium channels, Na(+)/K(+)-ATPase, and a long-opening cation channel. The cellular mechanisms underlying the circadian regulation of these ion channels and their functions in various tissues and organisms will also be discussed. Despite the magnitude of chronobiological studies in recent years, the circadian regulation of ion channels still remains largely unexplored. Through more investigation and understanding of the circadian regulation of ion channels, the future development of therapeutic strategies for the treatment of sleep disorders, cardiovascular diseases, and other illnesses linked to circadian misalignment will benefit.

Phosphatidylinositol 3 kinase-Akt signaling serves as a circadian output in the retina.

The daily rhythm of L-type voltage-gated calcium channels (L-VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L-VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor-induced ion channel trafficking and membrane insertion. Here, we report that PI3K-Akt signaling participates in the circadian phase-dependent modulation of L-VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L-VGCC current amplitudes and the expression of membrane-bound L-VGCCalpha1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L-VGCCalpha1D subunit compared with non-transfected photoreceptors. Interestingly, both PI3K-Akt and extracellular signal-related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L-VGCC rhythms. Inhibition of either pathway abolished the L-VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L-VGCC rhythms in retina photoreceptors.

Circadian controls outweigh acute illumination effects on the activity of extracellular signal-regulated kinase (ERK) in the retina.

Extracellular signal-regulated kinase (ERK) participates in numerous cellular functions including circadian-related activities. In the retina, the activity of ERK is under circadian control. However, it is not clear whether acute illumination changes or the circadian clocks in the retina have a larger impact on ERK activity, and the cellular distribution of activated ERK (pERK) as a function of circadian time in cone photoreceptors is not known. Chick embryos were exposed to the light or dark for various lengths of time after 12:12h light-dark (LD) cycles, or on the second day of constant darkness after LD entrainment. Retinas were excised after various exposure times and relative ERK activity was determined by western immunoblotting. We also performed immunohistochemical and immunocytochemical stainings on circadian entrained retina sections and dissociated retina cells. There is about a fourfold difference in ERK activity between retinas harvested at circadian time (CT) 4 and CT 16, and the internal circadian control of ERK activity in the retina overcomes external light exposure. Also, during the subjective night, pERK was more apparent in the outer segment of cones, while pERK distribution was more uniform throughout the photoreceptors during the subjective day. Our results imply that the activity of retinal ERK is influenced more by circadian oscillators than acute illumination changes. Hence, the circadian oscillators in retina photoreceptors play a major role in the regulation of photoreceptor physiology, which leads to the circadian control of light sensitivity in photoreceptors.

Interplay of nuclear factor-kappaB and B-myb in the negative regulation of androgen receptor expression by tumor necrosis factor alpha.

Increased androgen receptor (AR) levels are associated with prostate cancer progression to androgen independence and therapy resistance. Evidence has suggested that chronic inflammation is closely linked to various cancers including prostate cancer. Herein we show that the proinflammatory cytokine TNFalpha negatively regulates AR mRNA and protein expression and reduces androgen sensitivity in androgen-dependent LNCaP human prostate cancer cells. Decreased AR expression results from transcription repression involving essential in cis interaction of nuclear factor-kappaB (NF-kappaB) with the B-myb transcription factor at a composite genomic element in the 5'-untranslated region of AR. The negative regulation was abrogated when NF-kappaB activity was inhibited by a superrepressor of the inhibitory kappaB protein. In contrast, androgen-independent C4-2 (LNCaP-derived) cells fail to show AR down-regulation by TNFalpha, despite expression of B-myb and TNFalpha-induced NF-kappaB activity similar to that in LNCaP cells. The negatively regulated AR gene chromatin region showed TNFalpha-dependent enrichment of B-myb and the NF-kappaB proteins p65 and p50. In parallel, the histone deacetylase 1, corepressor silencing mediator of retinoid and thyroid hormone receptor and the corepressor-associated scaffold protein mSin3A were recruited to the inhibitory site. In C4-2 cells, neither NF-kappaB and B-myb, nor any of the corepressor components, were detected at the negative site in response to TNFalpha. Apoptosis was induced in TNFalpha-treated LNCaP cells, likely in part due to the down-regulation of AR. The androgen-independent, AR-expressing C4-2 and C4-2B (derived from C4-2) cells were resistant to TNFalpha-induced apoptosis. The results linking androgen dependence to the NF-kappaB and AR pathways may be insightful in identifying novel treatment targets for prostate cancer.

Melatonin Affects Mitochondrial Fission/Fusion Dynamics in the Diabetic Retina.

Mitochondrial fission and fusion are dependent on cellular nutritional states, and maintaining this dynamics is critical for the health of cells. Starvation triggers mitochondrial fusion to maintain bioenergetic efficiency, but during nutrient overloads (as with hyperglycemic conditions), fragmenting mitochondria is a way to store nutrients to avoid waste of energy. In addition to ATP production, mitochondria play an important role in buffering intracellular calcium (Ca2+). We found that in cultured 661W cells, a photoreceptor-derived cell line, hyperglycemic conditions triggered an increase of the expression of dynamin-related protein 1 (DRP1), a protein marker of mitochondrial fission, and a decrease of mitofusin 2 (MFN2), a protein for mitochondrial fusion. Further, these hyperglycemic cells also had decreased mitochondrial Ca2+ but increased cytosolic Ca2+. Treating these hyperglycemic cells with melatonin, a multifaceted antioxidant, averted hyperglycemia-altered mitochondrial fission-and-fusion dynamics and mitochondrial Ca2+ levels. To mimic how people most commonly take melatonin supplements, we gave melatonin to streptozotocin- (STZ-) induced type 1 diabetic mice by daily oral gavage and determined the effects of melatonin on diabetic eyes. We found that melatonin was not able to reverse the STZ-induced systemic hyperglycemic condition, but it prevented STZ-induced damage to the neural retina and retinal microvasculature. The beneficial effects of melatonin in the neural retina in part were through alleviating STZ-caused changes in mitochondrial dynamics and Ca2+ buffering.

Newly Identified Peptide, Peptide Lv, Promotes Pathological Angiogenesis.

Background We recently discovered a small endogenous peptide, peptide Lv, with the ability to activate vascular endothelial growth factor receptor 2 and its downstream signaling. As vascular endothelial growth factor through vascular endothelial growth factor receptor 2 contributes to normal development, vasodilation, angiogenesis, and pathogenesis of various diseases, we investigated the role of peptide Lv in vasodilation and developmental and pathological angiogenesis in this study. Methods and Results The endothelial cell proliferation, migration, and 3-dimensional sprouting assays were used to test the abilities of peptide Lv in angiogenesis in vitro. The chick chorioallantoic membranes and early postnatal mice were used to examine its impact on developmental angiogenesis. The oxygen-induced retinopathy and laser-induced choroidal neovascularization mouse models were used for in vivo pathological angiogenesis. The isolated porcine retinal and coronary arterioles were used for vasodilation assays. Peptide Lv elicited angiogenesis in vitro and in vivo. Peptide Lv and vascular endothelial growth factor acted synergistically in promoting endothelial cell proliferation. Peptide Lv-elicited vasodilation was not completely dependent on nitric oxide, indicating that peptide Lv had vascular endothelial growth factor receptor 2/nitric oxide-independent targets. An antibody against peptide Lv, anti-Lv, dampened vascular endothelial growth factor-elicited endothelial proliferation and laser-induced vascular leakage and choroidal neovascularization. While the pathological angiogenesis in mouse eyes with oxygen-induced retinopathy was enhanced by exogenous peptide Lv, anti-Lv dampened this process. Furthermore, deletion of peptide Lv in mice significantly decreased pathological neovascularization compared with their wild-type littermates. Conclusions These results demonstrate that peptide Lv plays a significant role in pathological angiogenesis but may be less critical during development. Peptide Lv is involved in pathological angiogenesis through vascular endothelial growth factor receptor 2-dependent and -independent pathways. As anti-Lv dampened the pathological angiogenesis in the eye, anti-Lv may have a therapeutic potential to treat pathological angiogenesis.

Circadian regulation of retinoschisin in the chick retina.

PURPOSE: To investigate the circadian regulation and acute illumination effects on the expression and secretion of retinoschisin from vertebrate retinas. METHODS: Retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12-hour light/12-hour dark (LD) cycles in ovo or in vitro. Quantitative real-time PCR and Western immunoblotting were used to examine the mRNA and protein expressions of retinoschisin at different circadian time points. Pharmacologic treatments in whole retina and dissociated retinal cell cultures were used to investigate the cellular mechanisms underlying the circadian regulation of retinoschisin content and secretion. Different illumination conditions were given to examine changes in retinoschisin content in association with acute light/dark adaptation. RESULTS: The mRNA level, protein expression, and secretion of retinoschisin were under circadian control, all of which were higher at night and lower during the day. The Ras, MAP kinase Erk, CaMKII pathway served as part of the circadian output regulating the rhythmicity of retinoschisin. Blockage of L-type VGCCs dampened the retinoschisin rhythm, but inhibition of L-type VGCCs did not completely abolish the secretion of retinoschisin. The protein expression of retinoschisin also responded to acute illumination changes. CONCLUSIONS: The mRNA and protein expression, as well as retinoschisin secretion, are under circadian control. L-type VGCCs play a role in the circadian regulation of retinoschisin, but the molecular mechanism underlying retinoschisin secretion does not depend on L-type VGCCs. Protein expression of retinoschisin in response to acute illumination changes depends on previous light exposure experience.

Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors.

Energy expenditure and metabolism in the vertebrate retina are under circadian control, as we previously reported that the overall retinal ATP content and various signaling molecules related to metabolism display daily or circadian rhythms. Changes in the fission and fusion process of mitochondria, the major organelles producing ATP, in retinal photoreceptors are largely dependent on light exposure, but whether mitochondrial dynamics in photoreceptors and retinal neurons are under circadian control is not clear. Herein, we investigated the possible roles of circadian oscillators in regulating mitochondrial dynamics, mitophagy, and redox states in the chicken retina and mammalian photoreceptors. After entrainment to 12:12-h light-dark (LD) cycles for several days followed by free-running in constant darkness (DD), chicken embryonic retinas and cone-derived 661W cells were collected in either LD or DD at 6 different zeitgeber time (ZT) or circadian time (CT) points. The protein expression of mitochondrial dynamin-related protein 1 (DRP1), mitofusin 2 (MFN2), and PTEN-induced putative kinase 1 (PINK1) displayed daily rhythms, but only DRP1 was under circadian control in the chicken retinas and cultured 661W cells. In addition, cultured chicken retinal cells responded to acute oxidative stress differently from 661W cells. Using pMitoTimer as a mitochondrial redox indicator, we found that the mitochondrial redox states were more affected by light exposure than regulated by circadian oscillators. Thus, this study demonstrates that the influence of cyclic lights might outweigh the circadian regulation of complex mitochondrial dynamics in light-sensing retinal cells.