In molecular biology laboratories, many tasks require fine motor control and high acuity vision. For example, lab technicians with visual impairment experience difficulty loading samples into the ...small wells of a horizontal agarose gel. We have developed a 3D-printable gel loading system which allows technicians with low-contrast vision to load gels correctly. It includes a casting tray, a bridge, and a modified comb. The system provides a high-contrast visual field to improve visibility, and the bridge allows pipette tips to be inserted at the correct location and only to the correct depth. The necessary computer files for printing this device are freely available to increase the accessibility of molecular biology laboratories to people with visual impairment.
We have produced a system for people with visual impairment to successfully load an agarose gel for gel electrophoresis. This system can be produced using a 3D printer. There are three components: a casting tray, a bridge, and a modified comb. The bridge fits into side notches in the casting tray and is suspended above the tray. The comb is inserted into the bridge while the molten gel is poured. When the gel cools, the comb is removed leaving the bridge in place. Pipettes can then only be inserted in the correct location and to the correct depth to load the wells. Importantly, the bridge and tray are contrasting colors to aid in visual discernment.
The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this ...entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength–sensitive cone pigment S-pigment or cone opsin (OPN1SW) nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.
Molecular mechanisms of the mammalian circadian clock have been studied primarily by genetic perturbation and behavioral analysis. Here, we used bioluminescence imaging to monitor
Per2 gene ...expression in tissues and cells from clock mutant mice. We discovered that
Per1 and
Cry1 are required for sustained rhythms in peripheral tissues and cells, and in neurons dissociated from the suprachiasmatic nuclei (SCN).
Per2 is also required for sustained rhythms, whereas
Cry2 and
Per3 deficiencies cause only period length defects. However, oscillator network interactions in the SCN can compensate for
Per1 or
Cry1 deficiency, preserving sustained rhythmicity in mutant SCN slices and behavior. Thus, behavior does not necessarily reflect cell-autonomous clock phenotypes. Our studies reveal previously unappreciated requirements for
Per1, Per2, and
Cry1 in sustaining cellular circadian rhythmicity and demonstrate that SCN intercellular coupling is essential not only to synchronize component cellular oscillators but also for robustness against genetic perturbations.
Vision is a highly rhythmic function adapted to the extensive changes in light intensity occurring over the 24-hour day. This adaptation relies on rhythms in cellular and molecular processes, which ...are orchestrated by a network of circadian clocks located within the retina and in the eye, synchronized to the day/night cycle and which, together, fine-tune detection and processing of light information over the 24-hour period and ensure retinal homeostasis. Systematic or high throughput studies revealed a series of genes rhythmically expressed in the retina, pointing at specific functions or pathways under circadian control. Conversely, knockout studies demonstrated that the circadian clock regulates retinal processing of light information. In addition, recent data revealed that it also plays a role in development as well as in aging of the retina. Regarding synchronization by the light/dark cycle, the retina displays the unique property of bringing together light sensitivity, clock machinery, and a wide range of rhythmic outputs. Melatonin and dopamine play a particular role in this system, being both outputs and inputs for clocks. The retinal cellular complexity suggests that mechanisms of regulation by light are diverse and intricate. In the context of the whole eye, the retina looks like a major determinant of phase resetting for other tissues such as the retinal pigmented epithelium or cornea. Understanding the pathways linking the cell-specific molecular machineries to their cognate outputs will be one of the major challenges for the future.
The molecular clockwork of mammalian cells Yi, Jonathan S.; Díaz, Nicolás M.; D’Souza, Shane ...
Seminars in cell & developmental biology,
06/2022, Volume:
126
Journal Article
Peer reviewed
Open access
Most organisms contain self-sustained circadian clocks. These clocks can be synchronized by environmental stimuli, but can also oscillate indefinitely in isolation. In mammals this is true at the ...molecular level for the majority of cell types that have been examined. A core set of “clock genes” form a transcriptional/translational feedback loop (TTFL) which repeats with a period of approximately 24 h. The exact mechanism of the TTFL differs slightly in various cell types, but all involve similar family members of the core cohort of clock genes. The clock has many outputs which are unique for different tissues. Cells in diverse tissues will convert the timing signals provided by the TTFL into uniquely orchestrated transcriptional oscillations of many clock-controlled genes and cellular processes.
Animals have evolved light-sensitive G protein–coupled receptors, known as opsins, to detect coherent and ambient light for visual and nonvisual functions. These opsins have evolved to satisfy the ...particular lighting niches of the organisms that express them. While many unique patterns of evolution have been identified in mammals for rod and cone opsins, far less is known about the atypical mammalian opsins. Using genomic data from over 400 mammalian species from 22 orders, unique patterns of evolution for each mammalian opsins were identified, including photoisomerases, RGR-opsin (RGR) and peropsin (RRH), as well as atypical opsins, encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5). The results demonstrate that OPN5 and rhodopsin show extreme conservation across all mammalian lineages. The cone opsins, SWS1 and LWS, and the nonvisual opsins, OPN3 and RRH, demonstrate a moderate degree of sequence conservation relative to other opsins, with some instances of lineage-specific gene loss. Finally, the photoisomerase, RGR, and the best-studied atypical opsin, OPN4, have high sequence diversity within mammals. These conservation patterns are maintained in human populations. Importantly, all mammalian opsins retain key amino acid residues important for conjugation to retinal-based chromophores, permitting light sensitivity. These patterns of evolution are discussed along with known functions of each atypical opsin, such as in circadian or metabolic physiology, to provide insight into the observed patterns of evolutionary constraint.
Synchronization of the mammalian master circadian pacemaker to the daily light/dark cycle is mediated exclusively through retinal photoreceptors. The mammalian retina itself is also a self-sustained ...circadian oscillator. Here we report that the retinal molecular circadian clock can be entrained by lighting cycles in vitro, but that rods, cones, and melanopsin (Opn4) are not required for this entrainment. In vivo, retinas of Opn4 ⁻/⁻; rd1/rd1 mice synchronize to light/dark cycles regardless of the phase of the master circadian pacemakers of the suprachiasmatic nuclei or the behavior of the animal. These data demonstrate that the retina uses a separate mechanism for local entrainment of its circadian clock than for entrainment of organism-level rhythmicity.
Mammalian circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), and current dogma holds that the SCN is required for the expression of circadian rhythms in peripheral tissues. Using a ...PERIOD2::LUCIFERASE fusion protein as a real-time reporter of circadian dynamics in mice, we report that, contrary to previous work, peripheral tissues are capable of self-sustained circadian oscillations for > 20 cycles in isolation. In addition, peripheral organs expressed tissue-specific differences in circadian period and phase. Surprisingly, lesions of the SCN in mPer2Luciferase knockin mice did not abolish circadian rhythms in peripheral tissues, but instead caused phase desynchrony among the tissues of individual animals and from animal to animal. These results demonstrate that peripheral tissues express self-sustained, rather than damped, circadian oscillations and suggest the existence of organ-specific synchronizers of circadian rhythms at the cell and tissue level.
The presence of an endogenous circadian clock within most mammalian cells is associated with the amazing observation that within a given tissue, these clocks are largely in synchrony with each other. ...Different tissues use a variety of systemic or environmental cues to precisely coordinate the phase of these clocks. The cornea is a unique tissue in that it is largely isolated from the direct blood supply that most tissues experience, it is transparent to visible light, and it is exposed directly to environmental light and temperature. Melatonin is a hormone that has been implicated in regulation of the cornea’s circadian clocks. Here, we analyze the ability of rhythmic melatonin to entrain corneas ex vivo, and analyze the phase of corneal circadian clocks in vivo both in light: dark cycles and in constant darkness. We find that the presence of a retina from a melatonin-proficient mouse strain, C3Sn, can photoentrain the circadian clocks of a co-cultured mouse cornea, but a retina from a melatonin-deficient strain, C57Bl/6, cannot. Furthermore, pharmacologic blockade of melatonin or use of a retina with advanced retinal degeneration, Pde6brd1, blocks the photoentraining effect. Corneal circadian clocks in vivo adopt an advanced phase in C3Sn mice compared with C57Bl/6, but the circadian clocks in the liver are unaffected. This observation is not attributable to a shorter endogenous period of the cornea or behavior between the strains. Some transcripts of circadian genes in the corneas of C3Sn mice also show an advanced phase of expression in a light: dark cycle, while the transcript of Per2 exhibits a light-dependent transient induction at the onset of darkness. We conclude that melatonin acts as a phase modifying factor in a rhythmic manner for the circadian clocks of the cornea.
Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the ...circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
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•Adipocytes express encephalopsin (OPN3), a 480 nm blue-light-sensitive opsin•Mice lacking OPN3 or blue light have diminished thermogenesis during cold exposure•Loss of OPN3 reduces oxygen consumption and energy expenditure•White adipocyte OPN3 promotes lipolysis during cold exposure
White adipocytes activate the lipolysis pathway to produce the free fatty acids that are used as heating fuel by brown adipose tissue. Nayak et al. show that Opsin 3 is required for blue-light-enhanced activation of the lipolysis pathway. This explains the low body temperature of Opn3 mutant mice.
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