Exposure of cells to visible light in nature or in fluorescence microscopy often is considered to be relatively innocuous. However, using the yeast respiratory oscillation (YRO) as a sensitive measurement of metabolism, we find that non-UV visible light has a significant impact on yeast metabolism. Blue/green wavelengths of visible light shorten the period and dampen the amplitude of the YRO, which is an ultradian rhythm of cell metabolism and transcription. The wavelengths of light that have the greatest effect coincide with the peak absorption regions of cytochromes. Moreover, treating yeast with the electron transport inhibitor sodium azide has similar effects on the YRO as visible light. Because impairment of respiration by light would change several state variables believed to play vital roles in the YRO (e.g., oxygen tension and ATP levels), we tested oxygen's role in YRO stability and found that externally induced oxygen depletion can reset the phase of the oscillation, demonstrating that respiratory capacity plays a role in the oscillation's period and phase. Light-induced damage to the cytochromes also produces reactive oxygen species that up-regulate the oxidative stress response gene TRX2 that is involved in pathways that enable sustained growth in bright visible light. Therefore, visible light can modulate cellular rhythmicity and metabolism through unexpectedly photosensitive pathways.M any organisms are exposed to visible light in their environment. Full sunlight can deliver up to 10 quadrillion photons of visible light·cm −2 ·s −1 (i.e., 2,000 μEinsteins·m −2 ·s −1 ), and cloud cover reduces this exposure only by a factor of 10. Even though sunlight provides photosynthetic energy to plants and a medium for the vision of many animal species, its highintensity rays damage living cells when light-absorbing molecules cannot safely disperse the energy that photons bring. Although the destructive capacity of UV light is widely appreciated, photons of visible light can be deleterious, e.g., by destroying cytochromes and thus affecting cellular respiration (1) or by producing reactive oxygen species (ROS) that cause damage to DNA, membranes, and other cellular components (2). To cope with the damaging effects of light, organisms have evolved different strategies ranging from the expression of shielding pigments, such as melanin and carotenoids (3), to active mechanisms that sense light and respond quickly to mitigate/repair damage, such as iris constriction to protect the retina (4, 5), light-avoidance movements by chloroplasts and mitochondria (6, 7), and the induction/activation of DNA photolyase (8). A third strategy to minimize damage from light is to anticipate and prepare for its effects through the use of cellular timing mechanisms such as a circadian clock (9).The budding yeast Saccharomyces cerevisiae lacks the wellcharacterized photoreceptors of many other organisms [e.g., cryptochromes, phytochromes, and the photoreceptors whitecollar 1 (WC-1), and rhodopsins] that sense light intensity and spe...

The use of luciferase reporters has become a precise, noninvasive, high-throughput method for real-time monitoring of promoter activity in living cells, especially for rhythmic biological processes such as circadian rhythms. We developed a destabilized firefly luciferase as a reporter for rhythmic promoter activity in both the cell division and respiratory cycles of the budding yeast Saccharomyces cerevisiae in which real-time luminescence reporters have not been previously applied. The continuous output of light from luciferase reporters allowed us to explore the relationship between the cell division cycle and the yeast respiratory oscillation, including the observation of responses to chemicals that cause phase shifting of the respiratory oscillations. Destabilized firefly luciferase is a good reporter of cell cycle position in synchronized or partially synchronized yeast cultures, in both batch and continuous cultures. In addition, the oxygen dependence of luciferase can be used under certain conditions as a genetically encodable oxygen monitor. Finally, we use this reporter to show that there is a direct correlation between premature induction of cell division and phase resetting of the respiratory oscillation under the continuous culture conditions tested.circadian rhythms ͉ luciferase reporter ͉ metabolic cycle ͉ Saccharomyces ͉ ultradian rhythms

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H+ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.

We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

Four decades of work have clearly established the existence of autonomous oscillations in budding yeast culture across a range of operational parameters and in a few strains. Autonomous oscillations impact substrate conversion to biomass and products. Relatively little work has been done to quantify yield in this case. We have analyzed the yield of autonomously oscillating systems, grown under different conditions, and demonstrate that it too oscillates. Using experimental data and mathematical models of yeast growth and division, we demonstrate strategies to increase the efficient recovery of products. The analysis makes advantage of the population structure and synchrony of the system and our ability to target production within the cell cycle. While oscillatory phenomena in culture have generally been regarded with trepidation in the engineering art of bioprocess control, our results provide further evidence that autonomously oscillating systems can be a powerful tool, rather than an obstruction.

A defining characteristic of embryonic cells is their ability to divide rapidly, even in tissues such as cardiac muscle, which cannot divide once fully differentiated. This suggests that regulators of cell division differ in embryonic and differentiated cells. LEK1 is a member of an emerging family of proteins with diverse functions but shared structural domains, including numerous leucine zippers, a nuclear localization site, and a functional Rb-binding domain. LEK1 is expressed ubiquitously in the developing mouse embryo from the earliest stages of differentiation through birth. It is absent in adult tissues, even those that maintain active cell division. We hypothesize that LEK1 is a regulator of mitosis restricted to the developing embryo and early neonate. Here, using BrdU incorporation, we show that LEK1 protein downregulation in cardiac myocytes correlates directly with cessation of DNA synthesis between neonatal days 6 and 10. In contrast, in an immortalized cardiac cell line (HL1 cells), both BrdU incorporation and LEK1 protein expression persist, and actively dividing cells express LEK1. However, BrdU incorporation can be decreased in these cells by treatment with a morpholino targeting LEK1 mRNA. These data suggest a role for LEK1 in regulating the normal embryonic cardiomyocyte cell cycle and in promoting continued mitosis in transformed, abnormally dividing cardiomyocytes.

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