scholarly journals Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

2019 ◽  
Author(s):  
Eric Poliner ◽  
Cameron Cummings ◽  
Linsey Newton ◽  
Eva M. Farré
Keyword(s):  
Cell Division ◽  
Circadian Rhythms ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Firefly Luciferase ◽  
Constant Light ◽  
Future Studies ◽  
Circadian Oscillators ◽  
Free Running

SummaryCircadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions, however, rhythms dampened within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, N. oceanica CCMP1779 and N. salina CCMP537. The reporter lines express free-running bioluminescence rhythms with periods of ~21-31 h that dampen within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are regulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.Significance statementStramenopiles are a large and diverse line of eukaryotes in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have developed bioluminescence reporter lines in Nannochloropsis species and provide evidence for the presence of a circadian oscillator in stramenopiles; these lines will serve as tools for future studies to uncover the molecular mechanisms of circadian oscillations in these species.

scholarly journals The Cell Division Cycle of Euglena gracilis Indicates That the Level of Circadian Plasticity to the External Light Regime Changes in Prolonged-Stationary Cultures

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1475
Author(s):  
Shota Kato ◽  
Hong Gil Nam
Keyword(s):  
Cell Division ◽  
Circadian Rhythms ◽  
Euglena Gracilis ◽  
Cell Cycle Progression ◽  
Light Signal ◽  
Cell Division Cycle ◽  
Regime Changes ◽  
Fitness Advantage ◽  
Subjective Night ◽  
Free Running

In unicellular photosynthetic organisms, circadian rhythm is tightly linked to gating of cell cycle progression, and is entrained by light signal. As several organisms obtain a fitness advantage when the external light/dark cycle matches their endogenous period, and aging alters circadian rhythms, senescence phenotypes of the microalga Euglena gracilis of different culture ages were characterized with respect to the cell division cycle. We report here the effects of prolonged-stationary-phase conditions on the cell division cycles of E. gracilis under non-24-h light/dark cycles (T-cycles). Under T-cycles, cells established from 1-month-old and 2-month-old cultures produced lower cell concentrations after cultivation in the fresh medium than cells from 1-week-old culture. This decrease was not due to higher concentrations of dead cells in the populations, suggesting that cells of different culture ages differ in their capacity for cell division. Cells from 1-week-old cultures had a shorter circadian period of their cell division cycle under shortened T-cycles than aged cells. When algae were transferred to free-running conditions after entrainment to shortened T-cycles, the young cells showed the peak growth rate at a time corresponding to the first subjective night, but the aged cells did not. This suggests that circadian rhythms are more plastic in younger E. gracilis cells.

scholarly journals The lysine demethylase dKDM2 is non-essential for viability, but regulates circadian rhythms in Drosophila

2018 ◽  
Author(s):  
Yani Zheng ◽  
Yongbo Xue ◽  
Xingjie Ren ◽  
Xiao-Jun Xie ◽  
Mengmeng Liu ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Histone Methylation ◽  
Molecular Mechanisms ◽  
Abnormal Development ◽  
Null Alleles ◽  
Constant Darkness ◽  
Post Translational Modification ◽  
Developmental Defects ◽  
Chromatin Dynamics

AbstractPost-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive, depending on specific pathological contexts. However, the role of KDM2 proteins during development in the whole organisms remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately one hour shorter than the control. Interestingly, opposite defects are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of how dKDM2 and its orthologs in vertebrates regulate circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.

Dissociation of motor activity circadian rhythm in rats after exposure to LD cycles of 4-h period

1997 ◽  
Vol 272 (1) ◽  
pp. R95-R102 ◽  
Author(s):  
J. Vilaplana ◽  
T. Cambras ◽  
A. Diez-Noguera
Keyword(s):  
Motor Activity ◽  
Activity Rhythm ◽  
Bright Light ◽  
Constant Light ◽  
Constant Darkness ◽  
Circadian Oscillators ◽  
Entrainment Process ◽  
Free Running ◽  
Anticipatory Activity ◽  
Degree Of Coupling

For > 30 days Wistar rats were subjected to six dark pulses per day (T4 cycles; 3 h light, 1 h dark) to study the possibility of dissociating their motor activity rhythm into distinct circadian components. Rats of both sexes were used, one-half of which were pinealectomized to examine the effect of the pineal gland on the entrainment process. Results show that when rats were maintained under T4 a 4-h rhythm in their motor activity was present. Rats showed anticipatory activity to dark phases, suggesting that the motor activity components are actually entrained to the external light/dark (LD) cycles. When rats were left under constant darkness after T4, some motor activity components coming from the dark phases free ran for several days with different circadian periods. This suggests that the motor activity pattern is generated by several circadian oscillators. Moreover, the free-running components of motor activity after T4 were more evident when T4 was applied after exposure to constant light than after exposure to constant darkness. These results support the hypothesis that the circadian system of the rat is formed by several circadian oscillators, whose degree of coupling depends on light conditions. In constant light, bright light may inhibit internal coupling within the system, making it subsequently more susceptible to the T4 cycles. No differences were observed between pinealectomized and sham-operated animals, although females were more sensitive to T4 cycles than males.

Continuous light or darkness and circadian periodic mitosis and metabolism in C and D8 mice

1961 ◽  
Vol 201 (2) ◽  
pp. 227-230 ◽  
Author(s):  
Franz Halberg ◽  
Cyrus P. Barnum
Keyword(s):  
Cell Division ◽  
Circadian Rhythms ◽  
Deoxyribonucleic Acid ◽  
Acid Metabolism ◽  
Specific Activity ◽  
Continuous Light ◽  
Liver Parenchyma ◽  
Constant Light ◽  
Constant Darkness ◽  
Spot Check

In immature C mice exposed first to alternating 12 hr of light and 12 hr of darkness (LD), and maintained thereafter in constant darkness for several days, the circadian rhythms in hepatic and pinnal mitosis are demonstrable by spot checks made at the approximate times of LD-synchronized peak and trough. Spot checks at same times in mice of same stock and age, kept for several days in constant light, reveal the cell division rhythm of liver parenchyma, but not that of pinnal epidermis. In immature D8 mice kept for several days in constant darkness, rhythms in hepatic mitosis, phospholipid, ribonucleic and deoxyribonucleic acid metabolism persist, while cell division rhythm in ear pinna of same animals is not detectable with the particular spot check used. In mice of same stock and age, on the 4th day in constant light, a significant rhythm persists in the relative specific activity of the hepatic phospholipid; timing of this metabolic cellular rhythm is drastically desynchronized from the reference standard of a 24-hr clock. Data reveal persistence of some mitotic and metabolic circadian rhythms under conditions studied, with phase drifts or phase shifts of these rhythms occurring both in relation to the 24-hr clock and among the rhythms themselves. These changes in external and internal timing of a circadian system are more extensive in constant light than in constant darkness.

scholarly journals Is the cell division cycle gated by a circadian clock? The case of Chlamydomonas reinhardtii.

1995 ◽  
Vol 129 (4) ◽  
pp. 1061-1069 ◽  
Author(s):  
K Goto ◽  
C H Johnson
Keyword(s):  
Cell Division ◽  
Circadian Clock ◽  
Chlamydomonas Reinhardtii ◽  
Cell Division Cycle ◽  
Circadian Rhythmicity ◽  
Circadian Period ◽  
Circadian Oscillators ◽  
A Cell ◽  
Free Running

Circadian oscillators are known to regulate the timing of cell division in many organisms. In the case of Chlamydomonas reinhardtii, however, this conclusion has been challenged by several investigators. We have reexamined this issue and find that the division behavior of Chlamydomonas meets all the criteria for circadian rhythmicity: persistence of a cell division rhythm (a) with a period of approximately 24 h under free-running conditions, (b) that is temperature compensated, and (c) which can entrain to light/dark signals. In addition, a mutation that lengthens the circadian period of the phototactic rhythm similarly affects the cell division rhythm. We conclude that a circadian mechanism determines the timing of cell division in Chlamydomonas reinhardtii.

scholarly journals The Circadian Protein PER1 Modulates the Cellular Response to Anticancer Treatments

2021 ◽  
Vol 22 (6) ◽  
pp. 2974
Author(s):  
Marina Maria Bellet ◽  
Claudia Stincardini ◽  
Claudio Costantini ◽  
Marco Gargaro ◽  
Stefania Pieroni ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Cancer Cells ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Antitumor Agents ◽  
Pharmacological Therapy ◽  
Drug Induced

The circadian clock driven by the daily light–dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other’s expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.

scholarly journals Prevention of EloR/KhpA heterodimerization by introduction of site-specific amino acid substitutions renders the essential elongasome protein PBP2b redundant in Streptococcus pneumoniae

2018 ◽  
Author(s):  
Anja Ruud Winther ◽  
Morten Kjos ◽  
Gro Anita Stamsås ◽  
Leiv Sigve Håvarstein ◽  
Daniel Straume
Keyword(s):  
Cell Division ◽  
Cell Elongation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cell Region ◽  
Protein Protein Interaction

AbstractThe RNA binding proteins EloR and KhpA are important components of the regulatory network that controls and coordinates cell elongation and division in S. pneumoniae. Loss of either protein reduce cell length, and makes the essential elongasome proteins PBP2b and RodA dispensable. It has been shown previously in formaldehyde crosslinking experiments that EloR co-precipitates with KhpA, indicating that they form a complex in vivo. In the present study, we used 3D modeling and site directed mutagenesis in combination with protein crosslinking to further study the relationship between EloR and KhpA. Protein-protein interaction studies demonstrated that KhpA forms homodimers and that KhpA in addition binds strongly to the KH-II domain of EloR. Site directed mutagenesis identified isoleucine 61 (I61) as crucial for KhpA homodimerization. When substituting I61 with phenylalanine, KhpA lost the ability to homodimerize, while it still interacted strongly with EloR. In contrast, both homo- and heterodimerization were lost when I61 was substituted with tyrosine. By expressing these KhpA versions in S. pneumoniae, we were able to show that disruption of EloR/KhpA heterodimerization makes the elongasome redundant in S. pneumoniae. Of note, loss of KhpA homodimerization did not give rise to this phenotype, demonstrating that the EloR/KhpA complex is crucial for regulating the activity of the elongasome. In support of this conclusion, we found that localization of KhpA to the pneumococcal mid-cell region depends on its interaction with EloR. Furthermore, we found that the EloR/KhpA complex co-localizes with FtsZ throughout the cell cycle.ImportanceTo ensure correct cell division, bacteria need to monitor the progression of cell division and coordinate the activities of cell division proteins accordingly. Understanding the molecular mechanisms behind these regulatory systems is of high academic interest and might facilitate the development of new therapeutics and strategies to combat pathogens. EloR and KhpA form a heterodimer that is part of a signaling pathway controlling cell elongation in the human pathogen S. pneumoniae. Here we have identified amino acids that are crucial for EloR/KhpA heterodimerization, and demonstrated that disruption of the EloR/KhpA interaction renders the cells independent of a functional elongasome. Furthermore, we found the EloR/KhpA complex to co-localize with the division ring (FtsZ) during cell division.

scholarly journals Machine Learning and In-Vivo Expression Analyses Reveal Circadian Clock Features Predictive of Anxiety in Humans

2021 ◽  
Author(s):  
Aziz Zafar ◽  
Rebeccah Overton ◽  
Ziad Attia ◽  
Ahmet Ay ◽  
Krista Ingram
Keyword(s):  
Machine Learning ◽  
Circadian Rhythms ◽  
Circadian Clock ◽  
Clock Gene ◽  
Molecular Mechanisms ◽  
Clock Genes ◽  
Anxiety Symptoms ◽  
Circadian Phase ◽  
Functional Studies

Abstract Mood disorders, including anxiety, are associated with disruptions in circadian rhythms and are linked to polymorphisms in circadian clock genes. Molecular mechanisms underlying these connections may be direct—via transcriptional activity of clock genes on downstream mood pathways in the brain, or indirect—via clock gene influences on the phase and amplitude of circadian rhythms which, in turn, modulate physiological processes influencing mood. Employing machine learning combined with statistical approaches, we explored clock genotype combinations that predict risk for anxiety symptoms in a deeply phenotyped population. We identified multiple novel circadian genotypes predictive of anxiety, with the PER3B-AG/CRY1-CG genotype being the strongest predictor of anxiety risk in males. Molecular chronotyping, using clock gene expression oscillations, revealed that advanced circadian phase and robust circadian amplitudes are associated with high levels of anxiety symptoms. Further analyses revealed that individuals with advanced phases and pronounced circadian misalignment were at higher risk for severe anxiety symptoms. Our results support both direct and indirect influences of clock gene variants on mood: while sex-specific clock genotype combinations predictive of anxiety symptoms suggest direct effects on mood pathways, the mediation of PER3B effects on anxiety via diurnal preference measures and the association of circadian phase with anxiety symptoms provide evidence for indirect effects of the molecular clockwork on mood. Unraveling the complex molecular mechanisms underlying the links between circadian physiology and mood is essential to identifying the core clock genes to target in future functional studies, thereby advancing the development of non-invasive treatments for anxiety-related disorders.

scholarly journals Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

2021 ◽  
Author(s):  
Margaret E Maes ◽  
Gabriele M Wögenstein ◽  
Gloria Colombo ◽  
Raquel Casado-Polanco ◽  
Sandra Siegert
Keyword(s):  
Environmental Changes ◽  
Genetic Material ◽  
Plexiform Layer ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Heparin Binding ◽  
Future Studies ◽  
Distinct Cell ◽  
Transgene Delivery

AbstractAdeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette.Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

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