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Ludwig Institute s Buzz Baum Discusses Drosophila RNAi Research

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At A Glance

Name: Buzz Baum

Position: Royal Society Research fellow, Ludwig Institute for Cancer Research, University College London branch

Background: Postdoc, Harvard Medical School — 1997-2001; PhD, genetics/molecular cell biology, Imperial Cancer Research Fund (now Cancer Research UK) — 1997; BA, biochemistry, Oxford University — 1993

After a brief period in the US, working in the lab of Harvard researcher Norbert Perrimon, Buzz Baum returned to his native England to continue the RNAi work he started as a postdoc. Recently, Baum spoke with RNAi News about his research.

How did you get started with RNA interference?

When I was doing my PhD work in Paul Nurse’s lab, I was working with yeast, but I was very interested in morphogenesis. In looking for a future path in science, I decided to go multi-cellular, and Drosophila is obviously a great organism. I wanted to do [in Drosophila] the genetics of cell biology, which was a very neglected field because it’s difficult to do fundamental cell biology in multi-cellular organisms.

But tools in flies are quite good, so I went to Norbert Perrimon’s lab, because Norbert had developed many of the key technologies that enable you to do cell biology in flies. So, you can make small clones of mutant tissue in otherwise normal animals to study the function of essential, fundamental, [and] cell-biological regulators.

I wanted to find the genes that regulate cell shape in flies. Although everyone [working in the field of] flies talks about how easy fly genetics is, I found it very difficult coming from yeast. In a way, taking the yeast perspective, I wanted tools that were much easier [in order] to study more genes. Having trained as a geneticist, you don’t want to study one or two genes — you want to do a survey to understand how a process is regulated before you choose the gene of interest.

There was a paper by [James] Clemens [of the University of Michigan, Ann Arbor] et al. in [the Proceedings of the National Academy of Sciences in] 2000, where they showed that RNAi could work in fly cell culture. I was already working with fly cells in culture because I could do movies of how cell shape changed over time by taking blood cells from the fly, so when RNAi came along, it was too good to be true for me. It was a tool where I could do large-scale genetic analysis of cell biological problems in a multi-cellular organism.

In fact, I ended up back using mostly single cells, so most of my lab now, and what I’ve been doing for the last few years, is mostly fly cell culture where we take primary cells from Drosophila and study the genes required for cell shape using RNAi as a tool to eliminate the messenger RNA to get a loss-of-function phenotype.

Can you talk about what you found [working in Perrimon’s lab]?

Basically, what happened was that another postdoc in the lab, Amy Kiger, and I worked to set up the high-throughput technology so that we could do high-throughput RNAi screens. From the very beginning, that’s what we wanted to do — being in a genetics lab where everybody does screens, our instinctive desire was to use RNAi to screen for gene function. What we did was adapt technology that Tim Mitchison’s lab at the ICCB were using across the quad in Harvard Medical School to do screens to find drugs affecting cell biological processes. We adapted that for RNAi.

We used two different Drosophila cell lines with different morphologies, and we tried to find the complement of genes that affect the shape of a round cell and a flat cell, trying to find which morphological regulators control the two different cell shapes. Obviously, many genes affect both cell types, such as genes required for cytokinesis, but other genes really are specific to cells with lamellipodia versus cells with … filopodia.

That’s what we’ve been doing, and recently we’ve done [a screen of] the … whole fly genome to find the genes controlling cell shape. We’ve gotten some very interesting, novel things that we’re studying.

We completed the screen awhile ago, [and] we’re going through the images manually, by eye. It takes a very long time — it’s quite hard to stay awake, often. We’re trying, using defined vocabulary, to give phenotypic descriptions of the phenotypes of every gene in the genome looking at actin, microtubules, and DNA. We come down to all the cell-cycle genes and other things, but we’re particularly interested in the morphological regulators.

We’ve just finishing screening by eye, and now we’re starting to validate our hits, and really work on those proteins and put them into pathways.

Do you have a time frame for this?

The wonderful thing about RNAi in Drosophila cell culture is that you can actually do a full-genome RNAi screen in a matter of weeks.

We did an automated microscopy screen, taking pictures of every single cell with double-stranded RNA targeting a single gene. You need automated microscopes, and that takes a few weeks to take the pictures. We just worked day and night, in shifts, standing by the microscope to be sure it didn’t crash taking all the pictures. Now, by eye, we’re going through all the images to annotate them. The whole project — if we did it properly, quickly, and as a priority — could [take] about three months to get the hits.

In fact, other screens … where you use luciferase-based readouts or other types of readouts … you can get results in a matter of days. It’s much more efficient in terms of time, but we wanted to do a really broad analysis and look at microtubules, actin, and DNA, so we could really see what was happening to the cells. Actually, that’s very useful background information because whenever anybody else does a screen, if they get a hit, and we look in our images and find that most of the cells are dead or cells are much bigger, then their hit may be a by-product of some other cell biological event. So, it’s quite a good background screen to have done to inform the results from other, more focused screens.

What about other projects, that you’re doing now or looking at?

Some [other] things that we’re doing that I think work really well with RNAi, which we’re quite excited about. [For example,] if you have a phosphorylated protein that you’re interested in, we have a kinase plate that my technician Veronica Dominguez in the lab makes. We have all these kinase plates and what we do is, if you have a phospho-antibody, you can just screen with that phospho-antibody on cells or in Westerns and identify the kinase involved — it takes no time at all. We’re doing that for a few phospho-antibodies and we’re trying to find kinases to hit those. You can then find phosphotases that act in opposition using the same kind of procedure.

The other thing we’re doing, which is very difficult to do in any other system except in yeast … is trying to do systematic modifier screens. What you do is you have a plate, for example of all the kinases from flies, then you take your gene of interest and you find the kinases and phosphotases [that] modify the phenotype. In that way, we’re placing genes into pathways. You can do that very systematically — with all the actin regulators we’ve identified, we want to put then into pathways with GTPases and kinases, and we can do that.

What about down the road?

The key [issue], which actually goes on from what Amy and I began, is: If you look at these pathway diagrams that people have on the walls of their offices, they have all these arrows linking genes. The fact is, that is sort of a curated analysis of thousands of papers done in hundreds of systems. What we can do in RNAi is take, for example in flies — we have many different cell lines in the labs and all of them are different — and ask: What kind of genes, genome-wide, are required for the functions, forms, and the behavior of different differentiated cell types?

By putting genes into pathways in the different cell types, we’re going to have a totally new perspective as to the way we look at cells because we’ve already found in the lab that there are some key actin regulators that really have much more fundamental roles in some cell types than in others, and maybe even in different pathways in different cell types.

That kind of analysis is very exciting — it’s sort of a systems biology approach.

You mentioned systems biology, which I would imagine would require a fairly extensive amount of collaboration. Are you working with anybody?

We have within the Ludwig [Institute] Marketa Zvelebil, a bioinformatician, and Ann Ridley, who works next door to me [and] is working on similar problems in mammalian cells. One thing we’re doing is building a database where we’re trying to cross-reference data from our RNAi screens with microarray data, as well as other databases. Marketa is helping build a database where we can put all our data and see it in the context of other information in the literature and out in the world.

I quite like the idea of doing small-scale systems biology. Often people talk about systems biology, but I think that [by] doing systematic loss-of-function analysis in different ways, we will learn things about how cells work in systems without having to do anything too ambitious with lots of people.

Have you ever thought of moving out of Drosophila into other organisms?

It’s a very good question and something we’ve thought a lot about. One of the problems we have is that it’s so easy to do the RNAi in fly cell culture. We can get lots of genes very quickly that we have lots of data on, but then to find out what they do in an animal — which is what you want — is much more difficult.

One thing we have thought about is C. elegans, where there are these feeding libraries available. I just went to a meeting where I met a lot of people who are doing genome-wide screens in C. elegans and I was actually very impressed. So, we have thought about that but we’re not doing it at the moment.

Obviously, mammalian cells are the future. For many, RNAi has changed the way we look at things now, because people can now, or very soon [will], be able to do these kinds of same studies in mammalian cells. In a way, that will take away a lot of the strength that these model organisms have had in the past.

I think most people are going to begin with collaboration, and with Ann Ridley’s lab next door, we hope to, in a collaborative way, start studying the genes we get and the pathways we identify in fly cells in human cells. Particularly, we’re trying to find the cells that affect motility in the system that Ann’s using.

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