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Illuminating Dark Mysteries of Intracellular Organelles

By Negin Martin
May 2010

statue depicting a hand
Lecturers in the Rodbell Lecture Seminar Series receive a statue depicting the hand of Nobel Laureate and former NIEHS Scientific Director Martin Rodbell, Ph.D., holding the three key elements involved in cell signaling. (Photo courtesy of Steve McCaw)

Jennifer Lippincott-Schwartz, Ph.D.
Lippincott-Schwartz engaged her audience as she answered a question. Her talk, the first Rodbell Lecture by a woman scientist, was postponed because of a blizzard in February and rescheduled during Women's History Month. (Photo courtesy of Steve McCaw)

Honored guest, Barbara Rodbell (center), Dr. Martin Rodbell's widow
An honored guest at each year's lecture, Rodbell's widow Barbara, center, sat with family friend Monroe Wechsler, Ph.D., left, of the North Carolina State University Department of Nuclear Engineering. Seated next to them is lecture host Birnbaumer, right, a former postdoctoral fellow in Rodbell's lab. (Photo courtesy of Steve McCaw)

Gary Bird, Ph.D.
Shown in the audience following his introduction of Lippincott-Schwarz, Bird appeared to ponder expanded applications for advanced photomicroscopy in his own work. (Photo courtesy of Steve McCaw)

David Miller Ph.D., left, and Jim Putney Ph.D., right
Also in the at-capacity audience were two NIEHS experts in the field of fluorescent microscopy - David Miller Ph.D., left, chief of the Laboratory of Toxicology and Pharmacology, and Jim Putney Ph.D., right, head of the Calcium Regulation Group. (Photo courtesy of Steve McCaw)

John Pritchard, Ph.D. presenting the Rodbell Award to Lippincott-Schwartz
As Scientific Director John Pritchard, Ph.D., presented the Rodbell Award to Lippincott-Schwartz, she joined the ranks of the leading scientists, including three Nobel Laureates, who have been so honored by the scientists at NIEHS. (Photo courtesy of Steve McCaw)

"Seeing is knowing," explained Jennifer Lippincott-Schwartz, Ph.D., at the beginning of the 12th annual Dr. Martin Rodbell Lecture Series Seminar on March 30 at NIEHS.

In her hour-long presentation, Lippincott-Schwartz dazzled her audience with a discussion of "Advances in Super-Resolution Imaging." In her talk, she demonstrated how new techniques of selective illumination are helping scientists see dynamic aspects of cell organelles that they could only have speculated about just a few years earlier - uncovering things she said "we'd never see with conventional fluorescent proteins."

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Lippincott-Schwartz( Exit NIEHS is the chief of the Section on Organelle Biology in the Cell Biology and Metabolism Branch at the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). She was elected in 2008 to the National Academy of Sciences (read profile( Exit NIEHS).

Tracking movement over time

Using super-resolution microscopy to study the eukaryotic endomembrane system, Lippincott-Schwartz has changed our understanding of many biological processes by revealing how intracellular membrane-bound organelles, such as endoplasmic reticulum (ER), Golgi apparatus, vesicles, lysosomes, and plasma membranes, work in concert to coordinate the dynamic movement of proteins and lipids throughout the cell.

The result of more than 35 years of development, the newest generations of fluorescent proteins are photo convertible moieties that can be turned on and off with a pulse of light, explained Lippincott-Schwartz - opening up for investigators "an incredible variety of approaches." In a 2002 study( Exit NIEHS, she and postdoctoral fellow George H. Patterson, Ph.D, reported on the discovery of a photoactivatable variant of avGFP, Aequorea victoria green fluorescent protein, (PA-GFP). The fluorescence of PA-GFP is increased 100 fold after irradiation with 413-nanometer light and it is stable for days, which allows scientists to track the movement over time of labeled proteins in a cell.

Lippincott-Schwartz gave several examples of types of biological questions that can be answered using photoactivated fluorescent proteins. She has used PA-fluorescent proteins to assess the inter-lysosomal exchange mechanism and to characterize the movement of vesicular stomatitis virus glycoprotein (VSVG)-PA-GFP between ER, Golgi apparatus, and plasma membrane. VSVG facilitates viral entry into cells.

In addition to dynamic applications, photoactivated fluorescent proteins can be used to measure protein turnover. Lippincott-Schwartz presented an elegant study comparing the ER-associated degradation of CD3-delta protein using either classical pulse chase experiments or PA-fluorescent proteins. Although both experiments revealed the same rate of turnover for the CD3-delta protein, the PA-fluorescent protein technology yielded many more data points over time - painting a more thorough picture of the dynamics of degradation.

Selective illumination enhances clarity

Photoactivated localization microscopy (PALM) is another application employed by Lippincott-Schwartz's lab to create more precise structural data. In this method, only a small fraction of PA-fluorescent labeled proteins become activated, and their movement is imaged repeatedly by PALM. Thousands of PALM images are analyzed and combined to create a detailed image. The PALM created image is clearer than the typically hazy images that result from overlapping GFP fluorescence. The photo gallery( Exit NIEHS at Lippincott-Schwartz's lab website features images, including ones of autophagosomes, ER-associated degradation, and Golgi apparatus organization.

Lippincott-Schwartz's lab has also helped in the development of a new imaging technology that produces the best resolution ever seen with an optical microscope. Interferometry is a technique used to study the pattern of interference created by two or more waves. Adapting interferometry to be compatible with photoactivated fluorescent proteins has given scientist a powerful imaging tool - interferometric PALM or iPALM. The new technology was first described in a research article( Exit NIEHS published in 2009 in Proceedings of the National Academy of Sciences accompanied by a number of high-resolution images of microtubules and focal adhesions.

Laboratory of Neurobiology Chief David Armstrong, Ph.D., and head of the Transmembrane Signaling Group, Lutz Birnbaumer, Ph.D., hosted the talk by Lippincott-Schwartz. NIEHS Calcium Regulation Group Staff Scientist Gary Bird, Ph.D., who nominated the speaker, gave her introduction.

(Negin Martin, Ph.D., is a biologist in the NIEHS Laboratory of Neurobiology Viral Vector Core Facility and a 2009 Science Communication Fellow with Environmental Health Sciences. She recently completed a postdoctoral fellowship with the NIEHS Membrane Signaling Group.)

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