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The busy life of the cell and its nucleus
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| David Stenoien, PhD |
The ability to view activities of genes and proteins within a single living cell begins with a jellyfish known as the Aequoria victoria, most often found in the waters of Pacific off the northwestern coast of the United States. This jelly fish is the source of a material called green fluorescent protein that has been used to mark the activities of proteins within cells, giving scientists a glimpse of the busy work of living cells.
Most high school biology students draw pictures of cells with the nucleus and various organelles depicted as stuck in place. Just as the drawing shows a static cell, that fixed image of a cell can remain in people’s minds.
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Mike Mancini, PhD |
“Now we know differently,” said David Stenoien, PhD, an instructor in Baylor College of Medicine’s department of molecular and cellular biology. Just as green fluorescent protein (GFP) marks the outlines of the jellyfish in the water, attaching it to proteins a scientist wants to study makes those proteins glow under a microscope.
With this relatively new molecular tool and a set of increasingly sophisticated microscopes, scientists in the darkened rooms of the department’s integrated microscopy core see pro teins within a living cell. This capability, when measured and evaluated, often turns preconceptions about the proteins being studied upside down.
| ER 40 movie | ||
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Using powerful microscopes and carefully prepared, live samples, Michael A. Mancini, PhD, an assistant professor in the department and director of the microscopy core, and his colleagues study how proteins regulate gene transcription, the ways in which proteins interact in real-time, how they are folded by the cell, and how proteins organize DNA, all crucial elements of cell life.
“Seeing is believing,” said Mancini.
Or is it? he hastens to add. Interpreting what is seen through the sophisticated microscopes in the microscopy core is an important part of the science going on in the laboratory every day. Research in his lab by Stenoien and others in recent years has focused upon looking at proteins where they actually work, in the cell, in real time.
Getting proteins to glow
GFP (green fluorescent protein) does not glow on its own, said Stenoien. “You have to provide it with energy that comes in the form of light.”
When light of a certain wavelength is shined on the protein to which GFP has been attached, “the atoms go to a more excited state or a higher energy level,” he said. “It needs to get back to the original energy level and as it does, it releases energy in the form of light.” With GFP, the light is green. A few color variants of GFP have been obtained through genetic engineering, allowing two or three proteins to be followed in the same cell. Since GFP was identified in the early 90’s, biotech companies have been scrounging the oceans for additional fluorescent proteins, and there are many new proteins (and variants) now commercially available; there could be dozens in the next year. New capabilities in the microscopy core can readily discern up to eight colors.
Researchers can now actually attach these different colored “GFP’s” (blue, green, yellow, and several different reds) to the many proteins they are interested in, enabling them to monitor different proteins in the cell at the same time.
“The technical problem now is centered on how to get a cell to express so many different proteins at the same time, in the right amounts. There has been little widespread appreciation of how overexpression wrecks the biology of the cell that people want to study,” said Mancini. Visualizing multiple proteins in different colors is an amazing new capability for any cell biologist, the stuff of pure wild imagination only a few years ago.
The fluorescent microscope
One of the key elements in this process is the fluorescent microscope with a light source that emits a broad spectrum of light. A filter is used to let only certain wavelengths of light into the microscope. The light at these wavelengths is focused on the specimen to be studied through a lens. When the proper wavelength of light hits the specimen, the fluorescent proteins attached to the cell’s protein begin to glow, making the protein glow as well.
The glow or emitted light goes back through the lens, which also has an emission filter that enables the appropriate image to be seen through the microscope’s eyepiece or by detectors that instantly create a digital image on the computer screen. A dichroic beam splitter attached to the microscope allows lights at the proper wavelength to pass through.
One downside of fluorescence microscopy is that only objects in one plane or at a single distance from the lens are in focus. “You focus on one object and the other is outside the focal point,” said Stenoien. “You see a sharp image where the object is in focus and a big blurry thing outside. You have to live with that with regular fluorescent microscopes.”
Confocal microscopes
One way to get around the focus issue is to use a laser scanning confocal microscope that substitutes a laser for the general light source.
“The laser is a very tight beam that scans across the sample, exciting one portion at a time,” said Stenoien. “Using the laser helps you focus on a single area.” The monochromatic nature of laser light avoids half of the normal filters.
To insure that the objects are in focus and eliminate extraneous light, a pinhole in front of the detector allows only light from the focal point to generate the digitized computer image. The tradeoff is between light and resolution, said Stenoien. A smaller pinhole will insure that the object and its surrounding are in sharp focus but it limits the amount of light that gets through.
“When we do live cell microscopy, we want to keep light levels as low as possible to avoid photo damage to the cells we are studying,” said Stenoien. “Usually with live cell microscopy, you sacrifice resolution.”
Multiple photon confocal microscope
One way to avoid the photo damage to live cells is to use two hits of light at a lower level. The two pulses of light must occur very quickly to bring the fluorophore or light-emitting part of the fluorescent protein to the necessary level of energy. The second pulse must occur within 10-16 seconds – a femptosecond, said Stenoien.
These pulses strike the in-focus plane of the specimen and enable scientists to use thicker section and lower energy light that can penetrate more easily, he said. Mancini is anxiously awaiting news on a funding request for $800,000 to get their first multiphoton microscope and for the core. There are only a couple of such scopes in Houston, which are not available in a multi-use facility.
Deconvolution microscopy and 3-D reconstruction
Another way to remove out-of-focus light from an image of a living cell is to use the deconvolution microscope. This microscope uses a computer to reassign light that is not in focus to its point of origin.
As the scientist focuses on a specimen, parts will be blurry and then become sharper at the focal point. “You still see a blurry image around the object,” said Stenoien.
The deconvolution microscope takes a series of images throughout the sample. Unlike the confocal that blocks vast amounts of out-of-focus light, based upon direct calculations and programming, the computer reassigns light from above and below the focal plane back to its origins.
“You are left with something that should look like the original objects,” said Stenoien. Since all the light is used and the cameras are very sensitive, these images are very bright and focused. There are always a trade-offs, and here computational time (10-30 minutes) and the need for thin samples, like cultured cells, are among those. Processing time continues to shrink with faster computers, with real-time deconvolution almost here, said Mancini.
What you see
The proteins on which much of the work involving single living cells concentrates are actually groups of protein molecules. “You can’t see a single molecule of a protein,” said Stenoien. “You could see 10 protein molecules. It depends on the sensitivity of the system.
Why live-cell imaging?
Those who study live cells under the microscope are trying to understand how different proteins and organelles in the cell behave under normal conditions. For example, tagging an enzyme called aurora kinase (the product of a gene found in breast cancer cells) with GFP allows the researcher to watch the enzyme go to the cell’s centrosome and follow that specialized organelle as the cell divides (or goes through mitosis). In collaborative studies, Stenoien, Mancini and Bill R. Brinkley, PhD, dean of the Graduate School of Biomedical Sciences at Baylor, performed real-time viewing that provided new information about the role of the centrosome in the development of many kinds of cancer.
FRAP (Fluorescence Recovery After Photobleaching)
| FRAP Example | ||
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One way of determining the mobility of proteins is to use a technique called Fluorescence Recovery After Photobleaching or FRAP. The microscopist uses a laser to repeatedly scan an area of the cell to “bleach” out the light that comes from GFP. That part of the protein takes a day or so to recover its ability to fluoresce. However, as the scientist watches, the “bleached” area recovers its ability to glow or fluoresce sometimes within seconds or many minutes. Molecules of protein move into the area, replacing the “bleached” molecules. By measuring the speed at which the glowing molecules replace the bleached ones, scientists can get a notion of the mobility of the proteins within the nucleus and its interactions with other proteins and cellular structures.
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Marilyn Mielke, D. Phil. |
A report by Mancini, Stenoien and Marilyn Mielke, D. Phil., in Nature Cell Biology last October described the nature of nuclear inclusions made of a mutant form of ataxin-1, a protein that causes the neurodegenerative disorder called spinal cerebellar ataxia-1. With all previous indications pointing to the disease version of ataxin-1 as a misfolded, “aggregated” protein, using FRAP, the three noted that the big blobs of protein occurred in two forms. One type, surprisingly, is very fluid and rich in proteasomes, large molecular “cleanup” machines. Another has high levels of ubiquitin, a short protein that marks other proteins for destruction. However, the second type of inclusion has a proteasome deficiency and is less mobile than the first.
| Fluid and Fusion | ||
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The first kind of inclusion “recovers” fluorescence quickly in a bleached area. In the second type, recovery is much slower.
“Although previous approaches suggested the inclusions were at
first thought to cause the disease, it appears that they may actually
be protective, at least at first, probably when they are highly mobile
and possessing functional proteasomes. The inclusions are probably a type
of cell-defense mechanism, when they are functioning,” Mancini said.
Mutant proteins that could cause disease are put into an inclusion and
chewed up, said Mancini. As he and his colleagues watched the inclusions,
they moved rapidly around inside the nucleus, already stuffed with 6-feet
of DNA. Inclusions that touched actually fused together. “How would
something 'aggregated' do that?” In individuals with the neurodegenerative
disorder, most cellular nuclei contain only one large inclusion. Mancini
theorizes that facilitating the mobility and metabolic capabilities of
the inclusions might affect the progression of the disease.
In a different set of studies from Mancini’s group, this time led by Mielke, a postdoctoral fellow, they are looking at the cellular localization and dynamics of two steroid receptor coactivators (SRC), SRC-1 and SRC-3. SRCs are key power boosters for steroid receptors and hormones can lead to their binding to receptors, like estrogen and the estrogen receptor (ER). Without estrogen, SRCs and ER are both nuclear and very unorganized, and very mobile. After adding estrogen or even some growth factors, the SRCs and ER rapidly interact. The big surprise is that the receptor and SRC “complexes” are much more dynamic than appreciated by standard approaches
“It’s real time biochemistry within the cell,” he said.
Mancini envisions single cell analysis as integrating the tools of molecular biology and biochemistry. Mielke, in a Saturday morning presentation before an animated group of molecular and cell biologists, said, “What is lagging are descriptive studies of the dynamics of complexes involved in processes such as transcription. We hope to be able, through our work, to shed some light on that.”
Descriptions of the movies.
ER 40 movie (back to
movie )
Steroid receptor on the move. A deconvolution microscope derived movie
of GFP-ER after adding estrogen. At the start, ER is very diffuse, even
glass-like, in the nucleus. After adding estrogen, within a few minutes,
reorganization takes place throughout the nucleus. These early events
are linked to transcription, but are observed long before routine assays
detect ER activity.
FRAP example (back
to movie)
Structural proteins can be dynamic in the nucleus. A classic nuclear structure
protein, NuMA (Nuclear and Mitotic
Apparatus) has long been studied biochemically (very insoluble).
A GFP-NuMA was examined by FRAP, and showed an amazing fluidity, with
recovery taking less than a minute, indicating a highly dynamic protein.
Fluid and fusion (back
to movie)
Ataxin-1 inclusions in the nucleus are not aggregated. This single nucleus
contains several GFP-ataxin-1 inclusions, and FRAP and time-lapse shows
them to be highly fluid, and they even move about and fuse to each other.
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