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Imaging a Hidden Universe | A Story of Stress Granules

Deborah January 10, 2014

 

Figure 1: Staufen protein (green) localizes to stress granules (lower image) in cells in response to incubation with arsenic (as)

Figure 1: Staufen protein (red) partially localizes to stress granules (lower image) with SG control protein G3BP (green) in cells in response to incubation with arsenite (AS). It is believed that stuafen participates in SG disassembly, while G3BP promotes assembly, which is consistent with the partial localization of staufen in the image above.  Images donated by N. Kedersha

Nancy Kedersha is a researcher based at Harvard Medical School and director of a confocal microscopy facility at the Brigham and Women´s Hospital in Boston, MA. A pioneer in the field of stress granule research, she was one of the very first to discover these dynamic structures, and to show that they form in all mammalian cells in response to environmental stress. “I first found stress granules the same way I find everything: By accident…” she quips honestly.

At the time Kedersha was working in the lab of Paul Anderson, were she still carries out active research; the lab was looking at the small, cytolytic granules seen in natural killer (NK) cells, as its primary research interest is the immune system’s role in inflammation and rheumatoid arthritis. In order to identify the protein contents of NK granules, the Anderson lab made monoclonal antibodies against proteins purified from them, but one of these antibodies – clone 2G9 – showed Kedersha something unexpected:

“[This antibody] actually recognized something else…it cross-reacted with an entirely different protein that is present in all cells—this different protein was cloned (using the antibody to screen an expression library) and it became known as TIA-1.” (TIA-1 is actually short for T-cell restricted intracellular antigen-1, but what’s in a name?)

Because she expected to see granules with the 2G9 antibody, she created conditions in which granules might form: “I tortured cells and suddenly saw the normally nuclear TIA-1 move into the cytoplasm and form granules.” Even when she exposed non-immune cell types to the stress stimulus – in this case heat shock – the normally nuclear TIA-1 moved into the cytoplasm to form these large granules. Kedersha then knew she was on the trail of something important.

The importance of these findings however, was met with a certain amount of resistance. Stress granules are transient, many of their components may only stay inside the granules for a few seconds and then move away; as a consequence they did not form a fixed structure that could be purified, which left some unconvinced that they were of any great significance in the early days of the field. However, Kedersha didn’t let this argument deter her: “Not being able to form a fixed structure doesn’t mean that [the proteins within stress granules] are not able to form several structures, dynamically. That means optic methods are the only way to get at [them].”

It was Kedersha’s growing affinity with the immunostaining technique that would help to settle things; it would also become her forte. She perfected an imaging technique used in staining and photographing cells in order to reveal their inner structures and organization. By “freezing” cellular contents using fixatives, and using antibodies to locate proteins post-mortem, many labs have now confirmed that hundreds of different proteins go to stress granules, some only in response to specific types of stress.

PIK-URB-KEAP-pFM

URB-KEAP-pFM

Figures 2 and 3: Some of Nancy Kedersha’s images featuring Proteintech antibodies (red). Other antibodies (green and blue) are from other vendors.  Figure 2 features anti-PIK3C2A (catalog no. 22028-1-AP). Figure 3 clearly shows nucleoli staining by URB1 (catalog no. 20023-1-AP). NB: images are not examples of stress granule formation.

Through her work she has amassed a huge portfolio of beautiful “bio art” images, which have featured in the likes of Discover and Time Magazine, as well as in many textbooks and museum exhibits. She has also won accolades for her photographic documentation of this hidden cellular micro-universe, including the most prestigious award in the field of scientific and medical photography: The Lennart Nilsson Award (presided over by the Karolinska Institute of Nobel fame).

In college, whilst not busy in the biology labs, Kedersha would spend her time backstage at University Theatre, managing and maintaining sets – backdrops, lighting, fittings and fixtures. Perhaps this experience was an early indication of a natural flair for immunofluorescence staining. She says of the technique now: “Using antibodies to light up cells is essentially just a more, highly technical way of lighting a theatrical production…the pictures of what antibodies let you see in cells are amazing.”

GB3P (green), Staufen (red) and FXR1 (blue) immunofluorescence staining. Extracted from an original figure donated by Nancy Kedersha comparing the staining after two diifferent fixation methods.

Figure 4: GB3P (green), staufen from Proteintech (red) and FXR1 (blue) immunofluorescence staining. Extracted from an original figure donated by Nancy Kedersha comparing antibody staining following two different fixation methods. GB3P and FXR1 antibodies are from other vendors.

Amazing is a fitting adjective for Kedersha’s images, from the examples in this post (all of them featuring at least one Proteintech antibody) you can see that as well as fulfilling a main objective – be that visualizing the localization of a specific protein or comparing fixation conditions – her images always tell a story. Take the images of staufen protein, a major component of stress granules and mRNA translation, and its localization with the mitotic spindle (figure 4); in some of the images you can clearly see the outline of chromosomes condensed and packaged, ready for the cells to progress through anaphase, if not in the early stages of it already. In another image (far right), two daughter cells can be seen in the last stages of cytokinesis, the remaining traces of staufen1 staining almost resembling arms outstretched in a final gesture of farewell.

Kedersha’s images are snapshots of life, as it happens; just on a smaller scale than most non-scientists are used to.  “They show that inner space is just as vast and as beautiful as outer space; each cell contains a galaxy of molecules in lieu of stars.”

Words By Deborah Grainger.

The author thanks Nancy Kedersha for her valuable input.

See a list of Proteintech’s Stress Granule related antibodies here

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