Interview with Jennifer Lippincott-Schwartz

Jennifer Lippincott-Schwartz

Let's start at the beginning, could you tell me what first inspired you to become a scientist?

As a young child I loved being outside exploring things. My father was a chemistry professor and when he took me to his lab, I remember thinking how fun it would be to be a scientist. I paid special attention to sciences in school. Nevertheless, in college, I majored in philosophy and psychology because at that point in my life I was questioning what knowledge is, what constitutes truth and how one tests different beliefs. In my later professional career as a scientist, this training influenced me greatly. After college, I went to Africa to be a high-school teacher and to experience the outside world. The teaching conditions were minimal in the rural school where I taught in Kenya. There were no textbooks, and the students came from farming families. These students were, nevertheless, bright and eager to learn the sciences I taught them. It was during this time that I recognized the power of science in changing our lives – from improving crops to providing cures for infections. By becoming a scientist, I realized I might be able to contribute to that process.

Can you tell us about your career path after your return to the United States?

When I came back to the United States, before starting graduate school in biology, I taught high school for another 2 years (teaching physics, chemistry, earth science and math) to cover expenses while my husband attended law school at Stanford University. I then entered the Master's degree program in biology at Stanford. At the time, Stanford was the center of the molecular biology revolution. My college education, just 4 years earlier, had taught me nothing about molecular biology, so it was a total shock to get immersed in this exploding field. Among my teachers were giants in the field, including Nobel Laureates Arthur Kornberg and Paul Berg. Being exposed to them and others was extremely impactful. I next entered the PhD program at Johns Hopkins University, where I was exposed to a different group of scientific giants – classical biochemists and geneticists. They were equally impressive. I setup projects using classical in vitro techniques but soon felt the need to see how the molecules being studied operated in the environment of an intact cell. I was fortunate, therefore, to join the lab of Douglas Fambrough at the Carnegie Institute of Embryology, which was associated with Hopkins. The lab was applying immunofluorescence microscopy using monoclonal antibodies tagged with fluorescent dyes to look at the subcellular distribution of different molecules. My PhD thesis characterized one of these monoclonal antibodies, which turned out to target LAMP1, a major lysosomal membrane protein. At the time, there were no labels for lysosomes other than dyes that became bright at low pH, so it was exciting to characterize LAMP1's biochemical and subcellular properties. My experience as a PhD student set the foundation for my long-term interest in understanding the relationships between different subcellular compartments, including how molecules circulate through these compartments and attain steady state distributions. My postdoc in Richard Klausner's lab at National Institutes of Health (NIH) in Bethesda followed this path. There, I was able to characterize a pathway by which unassembled protein subunits of receptors undergo degradation in the endoplasmic reticulum (ER), a process we now call ER-associated degradation. I also characterized how the Golgi undergoes complete disassembly and reassembly during treatment and washout with the drug brefeldin A. The latter findings were surprising to the field, as they implied a retrograde pathway from the Golgi back to the ER and questioned thinking in the field that organelles like the Golgi were stable compartments and unable to form de novo. Upon finishing my postdoc, I started my own lab at NIH with the aim of further studying organelle dynamics and membrane trafficking pathways in the secretory system.

What are the key moments in your research career so far?

I'll mention three key moments in my career that changed the way I thought and carried out science. The first moment came when my lab began tagging proteins with green fluorescent protein (GFP) to examine their distribution in cells. I never expected to see so much dynamism associated with subcellular structures, which included seeing tubules budding off organelles (including from the Golgi, lysosomes and endosomes), vesicles moving quickly along microtubules, and organelles breaking down and reassembling during mitosis. It was stunning to watch all these processes unfold in live cells, and the findings convinced me of the importance of studying them quantitatively using GFP.

A second key moment came when I realized that GFP could be used in FRAP experiments as a tool to uncover protein diffusion within cells. This discovery came serendipitously. I was testing how much laser power was required to photobleach GFP, since this would impact how long we could image our samples. I targeted a small square of the ER and exposed it to full laser power for a second to bleach the fluorescence in that area. The first image after photobleaching revealed a small black square, indicating the region of interest was successfully photobleached. Unexpectedly, however, when continuous images after the initial photobleaching were collected, I could see that fluorescence began recovering into the bleached region. It blew my mind to realize I was watching diffusion of unbleached molecules into the photobleached region. I had never thought about proteins moving freely by diffusion within an organelle or its implications, but that was what was occurring. The results changed my whole perspective, as they revealed that proteins within compartments are rapidly moving around rather than being fixed or anchored to some scaffold. Over the next few years, my lab set out to characterize this movement, developing various photobleaching strategies, including repeated photobleaching in one area to address whether molecules could freely diffuse between two areas of the cell. Then, a talented postdoc named George Patterson joined my group and developed photoactivatable GFP (PA-GFP), which is invisible until activated with a particular wavelength of light. Using PA-GFP, we could address even more questions related to how molecules circulate within cells by simply switching on molecules in one place and watching how this pool redistributed over time.

The third key moment in my career came when we were happily working with PA-GFP characterizing various intracellular trafficking pathways. A physicist by the name of Eric Betzig came to NIH one day and asked to chat with George and myself. Eric pitched the striking idea of using PA-GFP in a super-resolution approach involving building up an image through localization of thousands of isolated single molecules. At that point Eric didn't have a lab, nor the hands or reagents to test his idea, so I readily consented to his coming to my lab at NIH to try out the new approach. Harald Hess, a long-standing colleague of Eric's, along with Mike Davidson also joined the group. Once the microscope was set up and the PA-GFP-expressing cells put on it, we quickly got blinking molecules and began acquiring super-resolution images. It was unbelievably exciting. To get decent super-resolution images, the method (which we christened PALM for photoactivated localization microscopy) required that PA-GFP molecules be at a high enough density to identify the labeled structure. Microtubules and actin were ideal for imaging by PALM because of their regular shapes. For other organelles with irregular shapes, the approach required correlating the PALM image with an EM image to give cellular context. Suliana Manley in my lab then showed PALM could be used to explore the dynamism of individual proteins on membranes by tracking single PA-GFP tagged proteins in live cells. PALM, along with other super-resolution techniques developed around the same time, including STED, SIM and STORM, helped set off a revolution in super-resolution light microscopy, leading to the awarding of the Nobel Prize in Chemistry to Eric Betzig, Stefan Hell and W.E. Moerner.

Throughout your career, you've been at the forefront of developing and applying new technologies to questions in biology. Do you search for a technology to answer a question, or do you start with the technology and look at where it could be applied?

We do both. When I started my own lab, it was very clear to me that GFP was the solution for answering questions related to dynamism within cells. As one example, we had seen, using antibodies in fixed cells, evidence suggesting that Golgi enzymes were cycling through the ER but basically nobody believed it because the data was indirect. Using GFP in live cells allowed us to directly tackle this question, as well as others related to dynamic processes within cells. All the technology we developed related to GFP, such as FRAP, FLIP and PA-GFP, was toward the goal of studying cell dynamism. Even with PALM, which was a technique initially for fixed cells, we soon applied it to live cells to follow molecules by single-molecule tracking. Most recently, we've used high-resolution template matching in cryo-EM to line up different ribosomes in different elongation states to make a pseudo-time sequence movie of the whole translocation pathway. I look for new tools and techniques to see dynamism at different scales.

Looking into the future, do you see a new technology that you think will have a big impact in understanding cellular physiology?

It's hard to predict what future technology will pop up, because a lot of times it's unplanned. In terms of impacting cell physiology, tools that are likely to be especially helpful are probes for monitoring metabolites; for example, glucose, NADH, succinate and others. However, the extremely rapid fluxes of metabolites through metabolic pathways will likely make monitoring them difficult and mechanistic interpretations challenging. New approaches for both monitoring and analyzing fluxes will be needed.

Do you still get a chance to look down the microscope yourself?

Most of the time the images come to me in my office. I have a big computer monitor that people bring their movies to and we look at and discuss the movies together. But if we're starting off a new project, I like to see it under the microscope myself, to examine the whole field of view, not just examples that have been chosen. This is important to help a researcher decide what to look at in detail, find patterns and relationships, and to relate these to the rest of the sample. I think artificial intelligence might help us here.

Do you have any advice on how to go about building a collaborative network?

I've had many collaborators over my career, and the ones that have been most impactful are with people who are slightly outside my field. Early on, I had a strong collaboration with physicist Eric Siggia at Rockefeller University, who taught me a lot about soft matter physics and the role of flows and fluxes. These were crucial considerations in our studies of membrane trafficking and diffusion. With Eric Betzig, I learned a different physics domain, covering optics and engineering. Meeting these collaborators was not planned in any way. It is important to be extremely open-minded when you're interacting with other scientists. If you're going to a meeting, don't be afraid to go up to somebody and engage with them.

We're excited to hear your plenary lecture at our Biologists @ 100 conference, what are you looking forward to at the conference?

I'm looking forward to hearing all the different talks from different fields, which distinguishes this conference from other more focused meetings. I am also looking forward to meeting old colleagues as well as new people at the conference. Discussing the science that I will be presenting will also be rewarding.

Jennifer Lippincott-Schwartz was interviewed by Helen Zenner, Online Editor at Journal of Cell Science and Community Manager of FocalPlane. This piece has been edited and condensed with approval from the interviewee.