Welcome to an introductory video tutorial on cellular oscillators.
In this video, we will discuss what cellular oscillators are,
how they were discovered, and how these oscillators can be synchronized.
Molecular circadian clocks exist in nearly every cell in the body. To study circadian
rhythms at a cellular level, cell cultures are created by harvesting tissues from an organism.
This procedure isolates the cellular clocks from the influence of other organs or the environment.
In order to keep cells alive in a dish, they must be given proper nutrients,
including amino acids, vitamins, and growth factors. All of these essential components
together are called growth medium. It is also important that the cell culture
dishes are stored under proper conditions, where environmental factors are controlled.
Scientists can observe circadian rhythms in cells by measuring gene expression. There are
2 ways by which this is commonly done. One way is by the use of a reporter gene such as luciferase.
These assays allow for high-frequency sampling and are less labor intensive.
But this technique can only measure the activity of one gene at a time. For more information,
please see our tutorials on reporters and how they are used in circadian biology.
Another way of measuring gene expression is by mRNA quantification.
While this method is more labor intensive and cannot measure the same individual cell over time,
it can measure the activities of many, or even thousands, of genes at once,
and can be used without the need for genetic modification of the organism being studied.
mRNA quantification allows the researcher to take a snapshot of the mRNA content at one
point in time. To find a rhythm, a researcher will need multiple snapshots in a row -- for example,
every 2 hours, in different batches of cells-- to create a pattern of gene expression over time.
For the experiments we will review, the investigators created an RNA blot to measure
specific mRNAs in their sample at each time point. The more mRNA, the darker the band.
In this example, investigators cultured fibroblasts from rat skin cells
and collected samples every 4 hours. They measured mRNA for the clock gene Per2,
and for c-Fos, a gene that reflects cellular activation. They also measured a control gene
to make sure that they are measuring from the same amount of sample at each time point.
The results from this experiment showed that Per2 mRNA levels are constant,
that c-Fos is not expressed, and that the control gene is expressed at constant levels.
If the fibroblasts have a clock, we would expect to see cycling Per2 expression. Is
there some kind of timing signal missing in the culture that would be present in
the whole organism? If they are just missing a signal, what might that be?
In an animal, peripheral clocks are synchronized by the master clock of the body, the
suprachiasmatic nucleus, or SCN. The SCN receives light input and can adjust to
changes in daylength. It relays this information to other cells in the body, so that the body
clocks are aligned. How does the SCN send this signal? One way might be through the bloodstream.
Blood can be reduced to serum, which contains lipids, macromolecules, growth factors, hormones
and other nutrients. Scientists tested serum to determine if circadian cues are passed in blood.
Using a technique called a serum shock, cells are exposed to a high concentration of serum
to activate many cell signaling pathways.
The fibroblasts in our previous example were given a serum shock for 2 hours,
and then placed in a serum-free environment.
After the serum shock, Per2 began to display rhythmic expression with a peak every 24 hours.
This rhythm was not seen for cFos, which only showed a peak right after the serum shock, or
for the control gene, which maintained a constant level of expression throughout the experiment.
These data can be graphed to visualize patterns of expression. We just saw that the clock gene Per2
became rhythmic only after the serum shock. The constant expression prior to the serum shock
would be represented by a horizontal line on the graph. The rhythmic expression of Per2 and
other clock genes following the serum shock is represented by an oscillating curve on the graph,
with the peaks and troughs corresponding to high and low mRNA band intensity.
Taking into account that each single time point comes from a culture dish with tens of
thousands of cells, researchers came up with two different hypotheses to explain their results.
The first hypothesis, called induction, is the idea that individual cells are not rhythmic,
and only begin to cycle after their clocks are started by the serum shock.
The second hypothesis, called synchronization, is the idea that individual cells are cycling
all along, but are out of phase with one another. It is impossible to discriminate between these
hypotheses by studying mRNA levels from entire plates, because the overall mRNA signal would
be the same in both cases.population. To test these hypotheses, investigators
examined Per2 rhythmicity of individual cells using a luciferase reporter.
The investigators used fibroblasts cultured from per2 luciferase mice, which allows the
investigators to determine how much per2 is being made and when from individual cells.
The picture shows the rhythmicity of Per2 expression for two cells in the same
dish before serum shock and the sum of expression for 100 such individual cells. This finding
illustrates that individual cells have rhythmic Per2 expression before serum shock, and that the
desynchrony of the entire population causes their summed expression to appear constant.
Therefore, we can conclude that individual peripheral cells have cycling clock genes;
however, these cells are not synchronized in the absence of external cues.
This finding supports the synchronization hypothesis.
The many cells in a dish are oscillating independently, and are only synchronized after
some kind of manipulation, like the serum shock. These experiments demonstrate that serum has the
ability to synchronize cellular oscillators. But how? As we mentioned, serum contains a wide
range of components, many of which are unknown. To find a single component of serum that can reset
peripheral rhythms, researchers searched for a candidate that is secreted with a daily rhythm,
and has receptors in most peripheral cell types. An ideal candidate for one-way
communication from SCN to periphery would not have receptors in the SCN.
One class of hormones, called glucocorticoids, is present in serum and meets these criteria.
Therefore, it was hypothesized that glucocorticoids synchronize
peripheral oscillators within the body.
Researchers decided to examine whether an artificial glucocorticoid,
called dexamethasone, could synchronize cellular oscillators. Dexamethasone,
called dex, is more stable than endogenous glucocorticoids and therefore easier to handle.
In one study, the investigators treated cells with dex and measured clock gene
mRNA. They found that expression of several clock genes,
including Per2 and Cry1, began to show a rhythmic expression pattern after dex treatment.. Moreover,
expression of clock-controlled genes such as Rev-erbα also began to cycle.
When the researchers tried the same experiment with SCN tissue,
they discovered that dexamethasone impacts only the peripheral tissues, and not the SCN. Thus,
glucocorticoids are a potential way in which the SCN can synchronize peripheral clocks.
Another way the SCN might synchronize peripheral clocks is through temperature.
Most organisms have small changes in body temperature throughout the day,
controlled by the SCN. Researchers wanted to study if a temperature change is a
potential mechanism through which the SCN entrains peripheral oscillators.
One experiment studied the effects of temperature using the Per2 luciferase mice we discussed
before. The incubation temperature for fibroblasts was raised 6 degrees celsius for 30 minutes.
In response to this temperature shock, the cells became synchronized and showed a Per2 rhythm.
This result shows that a change in temperature has the ability to synchronize the clocks in cells,
just like dexamethasone and serum shock. A separate experiment tested whether
tissues can entrain to a repeating change in temperature, like what our bodies experience.
Tissue samples from the lung and SCN were all exposed to a temperature cycle,
where the temperature alternated just 1.5 degrees Celsius every 12 hours.
When they exposed the lung cultures to the temperature rhythm, shown in red,
they found that the Per2 rhythm in the cells aligned to the phase of the temperature rhythm.
Then, they exposed a second set of cultures to the same temperature cycle,
but 12 hours out of phase of the first example, shown in blue. They found that the second set
of cultures cycled in the opposite phase as the first set, corresponding to the temperature cycle.
In contrast, two sets of SCN cultures exposed to opposite temperature cycles
kept the same phase, and therefore were not entrained by the temperature cycles.
The dexamethasone and temperature treatments show that peripheral cells are sensitive to
different entraining signals than SCN cells. We still don’t know exactly how the SCN entrains
peripheral clocks, but glucocorticoids and temperature are both potential ways that
these peripheral clocks in our bodies are synchronized to the light cycle by the SCN.