Category Archives: stress response

Imaging translation of single mRNAs in live cells

Translating the information encoded in mRNAs into proteins is one of the most basic processes in biology. The mechanism requires a machinery (i.e. ribosomes) and components (mRNA template, charged tRNAs, regulatory factors, energy) that are shared by all organisms on Earth. We’ve learned a great deal about translation over the last century. We know how it works, how it is being regulated at many levels and under varuious conditions. We know the structures of the components. We have drugs that can inhibit translation. With the emergance of next-gen sequencing, we can now perform ribosome profiling and determine exatly which mRNAs are being translated, how many ribosomes occupay each mRNA species and where these ribosomes “sit” on the mRNA, on average. New biochemical approaches like SILAC and PUNCH-P can quantifiy newly synthesized proteins & peptides. Yet, all of that information comes from population studies, typically whole cell populations. Rarely, whole transcriptome/ribosome analysis of a single cell is performed. Still, there is no dynamic information of translation, since cells are fixed and/or lysed. And there is no spatial information regarding where in the cell translation occurs (poor spatial information can be determined if cell fractionation is performed, which is never a perfect separation of organelles/regions and we are still not at the stage of single organelle sequencing).

Imaging translation in single cells is intended to provide both spatial and dynamic information on translation at the single cell and, hopefully, single mRNA molecule resolution. Recently, four papers were published (on the same day!) providing information on translation of single mRNAs. Here is a summary of these papers.

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ASCB15 – part 2

I ended Part 1 after the morning session on pushing the boundaries of imaging.

After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.

After this session, I attended my first “discussion table”. Continue reading

The microscope’s light may affect your experiment

The conditions used for microscopy are often not “physiological” conditions. If we are talking about live imaging, then the cells are usually in culture, placed on a glass surface and grown in an artificial media. In many cases, we use genetically encoded fluorescent markers, that are rarely inert. These are acceptable and known limitations of the system.
However, when we think about microscopy, we do not often consider to effect of the light itself. The light we use can have deleterious effect. For instance, UV light is known to cause damage to cells, e.g. creating reactive oxygen species (ROS), creating thymidine dimers, cross-linking macromolecules and probably more. That is why people tend to limit the use of fluorescent proteins that are excited by UV light (such as blue fluorescent proteins or some photoactivated proteins).

Yet, visible light is considered relatively harmless. Now, a new study suggests that visible light, particularly blue-green light (which is used to excite green-yellow fluorescent proteins), can affect the metabolic state of the cell.  How?

Well, all cells contain light absorbing molecules. Some molecules function as light sensors or light energy harvesting molecules: Chlorophyll, cryptochromes, phytochromes, photoreceptors, rhodopsins, and fluorescent proteins. Other molecules absorb light, e.g. pigments.

However, some cells do not have obvious light sensitive molecules. For example, the budding yeast. In this paper, Carl H. Johnson’s lab looked at the effect of visible light on yeast respiratory oscillations (YRO) and oxidative stress. Apparently, under some conditions, yeast show 1-6hr oscillations of oxygen consumption and metabolite productions. surprisingly, shining white, blue or green light (but not red) has shortened the cycle  and decreased the amplitude of oxygen consumption. There was also increased expression of antioxidant enzymes, and increased light sensitivity to oxidative-stress mutants.

The effects of different spectra of light on the YRO. Oscillations were initiated in the dark until stable oscillations formed (black line, left y-axis). Then 12-h treatments of red, blue, or green light were administered (colored lines matching color of light, right y-axis) with 12 h of darkness between treatments. After the application of colored light, two 12-h white light treatments were given. Light intensities of each treatment are shown on the right y-axis and are indicated by numbers under each of the colored or gray lines showing light treatment. Source: Robertson J B et al. PNAS 2013;110:21130-21135

The effects of different spectra of light on the YRO. Oscillations were initiated in the dark until stable oscillations formed (black line, left y-axis). Then 12-h treatments of red, blue, or green light were administered (colored lines matching color of light, right y-axis) with 12 h of darkness between treatments. After the application of colored light, two 12-h white light treatments were given. Light intensities of each treatment are shown on the right y-axis and are indicated by numbers under each of the colored or gray lines showing light treatment. Source: Robertson J B et al. PNAS 2013;110:21130-21135

Based on several other assays, the authors suggests the blue-green light harms cytochromes in the respiratory electron transport process. Photoinhibition of the electron transport causes accumulation of ROS and oxidative stress response. It is hypothesized that the shortened oscillation periods and reduced amplitude of oxygen consumption (= less respiration) is a cellular mechanism to reduce the photoinhibition to electron transport, thus reduce oxidative damage.

What does this mean to microscopists?

It only suggests that long term exposure to light (either in prolonged live imaging, or just the incubation conditions) can affect the metabolic state of the cell and therefor may have either deleterious effect on the health of the cell, or create false positive or false negative results, particularly in experiments that are designed to study respiration and oxidative stress. Obviously, if you have light sensors or light harvesting molecules, their absorbance wavelengths need to be taken into account.

Practically, it is always a good advice to minimize the exposure to light (which we do anyway to reduce photobleaching).

ResearchBlogging.orgRobertson JB, Davis CR, & Johnson CH (2013). Visible light alters yeast metabolic rhythms by inhibiting respiration. Proceedings of the National Academy of Sciences of the United States of America, 110 (52), 21130-5 PMID: 24297928