An Interview with Dr. David Drubin | Biowire Spring 2012

Biowire, Spring 2012, 10–12

Biowire Spring 2012 — Live Cell Imaging of Signaling Pathways

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Dr. David Drubin - Professor of Cell and Developmental Biology at UC Berkeley

Dr. David Drubin – Professor of Cell and Developmental Biology at UC Berkeley

 

In this issue, our thought leader is Dr. David Drubin, Professor of Cell and Developmental Biology at UC Berkeley, whose recent live-cell imaging studies dramatically changed our understanding of clathrin-mediated endocytosis in mammalian cells1.

Clathrin-mediated endocytosis (CME) enables cells to take up nutrients and regulate the composition of cell-surface lipids and proteins, which in turn controls how the cell responds to environmental signals, differentiates, and grows. CME is the primary route for endocytosis, and alterations of the CME process are implicated in the pathogenesis of some cancers and atherosclerosis, as well as how viruses and harmful bacteria infect cells. David Drubin, Ph.D., Professor of Cell and Developmental Biology at the University of California, Berkeley, is using genome editing and high-throughput live-cell imaging to transform the conventional understanding of CME in mammalian cells.


Attracted to science at the cusp of the DNA and the cloning revolution that led to the startup of Genentech and other biotechs, Dr. Drubin’s graduate school rotations at UC San Francisco led him to his current research focus.

Dr. David Drubin:
My third rotation was with Marc Kirschner at UCSF. The first time I looked in the fluorescence microscope in his lab, I saw how beautiful the images of actin and microtubules were. There was no going back. I was going to be a cell biologist.

I now have a fairly large group at Berkeley, as well as an unusual situation. My wife, Georjana Barnes, whom I met during my post-doctoral studies at MIT, is also a professor at Berkeley. We combined our labs, which total 18 people. Many people are amazed that we do this and that it works. My wife and I each have different strengths as well as different things we like to do and don’t like to do. For people in the lab, this works out well. They will go to us for different kinds of questions.

Drs. Drubin and Barnes use real-time imaging techniques to study the molecular mechanisms of membrane trafficking and chromosome segregation events in live cells. A significant body of the duo’s work is in budding yeast. In recent years, Dr. Drubin began to translate his knowledge from yeast into mammalian cells, with surprising results.

Dr. David Drubin:
We noticed that endocytosis had been described differently in yeast and mammalian cells. In yeast, the steps of endocytosis occur in a regular, predictable order with high efficiency. In mammalian cells, the process was initially determined to be much less efficient and predictable. This didn’t seem right to us because the proteins involved were essentially the same in the two organisms.

It occurred to us that the way endocytosis had been studied was different between the yeast and mammalian cells. We theorized that the observed differences in the process might be attributable not to the actual intrinsic differences between the organisms, but rather to how the process had been studied.

In yeast, it is routine to integrate into the genome fluorescent protein tags, so the fusion proteins are expressed at their normal endogenous levels. In mammalian cells, this is not at all routine. People engineer a fusion gene in a test tube, and then they add that hybrid gene into the cells ectopically so the protein product is then over-expressed.

When people characterize the dynamics and the intracellular organization of proteins in mammalian cells, they are actually looking, not at the endogenous levels of proteins, but at the proteins expressed at higher levels than they are normally expressed in the cell. However, to my knowledge, the view that protein over-expression caused the observed differences between yeast and mammalian cells was not a view others had shared. Many people were invested in heterogeneity being real.

An ideal study of the mammalian CME process would require the expression of tagged proteins at their endogenous levels. However, a way to do so didn’t present itself until Fyodor Urnov, Ph.D., both a UCB colleague and a scientist at Sangamo Biosciences, suggested to Dr. Drubin that zinc finger nucleases (ZFNs) could target gene integrations in mammalian cells.

Dr. David Drubin:
ZFN technology could be used to do the same kind of moleculargenetic manipulations that we had done routinely in yeast. We set out to engineer a mammalian cell’s genome so that it would express fluorescent protein fusions at endogenous levels. At first, Sangamo was doing the genome editing. Then, Sangamo trained a few people in my lab, Jeff Doyon, Aaron Cheng, and Jackie Cheng. Our people actually became proficient at it.

 

"ZFN technology could be used to do the same kind of molecular-genetic manipulations
[in mammalian cells] that we had done routinely in yeast."
— Dr. David Drubin

 

When we tagged the endogenous clathrin light chain A and dynamin-2 genes with RFP and GFP using the zinc finger technology, we were amazed. The clathrin-mediated endocytosis process that we observed was much more efficient and regular than reported in the literature.

In prior studies in mammalian cells, the process was observed to be extremely heterogeneous, so much so that one couldn’t predict the order and timing of events. There was also significant heterogeneity in the size and shape of the endocytic structures in the mammalian cells. However, when we expressed the tagged proteins at endogenous levels, the process looked much more homogenous and similar to yeast than what had been described previously.

Cellular processes should be studied as close to their natural states as possible. I suspect that, as we see more uses of zinc finger nucleases [for tagging endogenous genes], people will find that they have been inadvertently perturbing the processes that they have been studying.

Tagging two endogenous genes, left under their natural promoters, opened up a data collection boon: unbiased high-throughput screening and analysis.

Dr. David Drubin:
The standard technique for expressing fluorescent proteins in mammalian cells is to transiently transfect the genes into the cells. Since different cells will take up different amounts of DNA, there is significant cell-to-cell variation in expression. Typically, people choose the cells that appear normal, and then image and study those cells. That introduces bias.

When we used the ZFNs to fluorescently tag endogenous genes, we produced a clonal cell line in which every cell is expressing the tagged proteins at endogenous levels. This feature allowed us to use software programs to image many different cells repeatedly in an unbiased manner. We did experiments in which we quantified tens of thousands of these dynamic events systematically in every single cell, without the scientist using his or her judgment to say the cell is fairly normal or well behaved.

Previously, it was difficult to predict how the CME process would change in response to a stimulus. Now, with a regular profile for the kinetics of the CME process, Dr. Drubin’s group is exploring how different cargos, stressors, and growth factors, among other things, affect CME.

Dr. David Drubin:
Endocytosis also plays different roles in different cell types. For example, in a nerve cell, endocytosis occurs very quickly every time an action potential is fired because the cell has to rapidly take up its neurotransmitters after they are released. We’ve been engineering stem cells that express fluorescently tagged endocytotic proteins at endogenous levels. The beauty of the stem cells is that we can differentiate them to become all sorts of different types of cells. We want to know what happens to the process as a cell differentiates into a nerve cell, a liver cell, or a heart cell — and how those changes alter the dynamics and regulation of endocytosis.

We are also examining the clathrin-mediated endocytosis process in finer detail. Using three more zinc fingers from Sigma®, we are adding markers that help reveal how different stages of the pathway are regulated.


 Reference

  1. Doyon JB, et al. Rapid and efficient clathrin-mediated endocytosis revealed in genomeedited mammalian cells. Nat Cell Biol. 2011;13:331–37.

 

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