Martin Chalfie: GFP and After

Chalfie is interested in sensory mechanotransduction—how are mechanical deformations of cells converted into chemical and electrical signals. Examples are touch, hearing, balance, and proprioception, and (hooray!) he references development: sidedness in mammals is defined by mechanical forces in early development. He studies this problem in C. elegans, in which 6 of 302 nerve cells detect touch. It’s easy to screen for mutants in touch pathways just by tickling animals and seeing if they move away. They’ve identified various genes, in particular a protein that’s involved in transducing touch into a cellular signal.

They’ve localized where this gene is expressed. Most of these techniques involved killing, fixing, and staining the animals. He was inspired by work of Shimomura, as described by Paul Brehm that showed that Aequorin + Ca++ + GFP produces light, and got in touch with Douglas Prasher, who was cloning GFP, and got to work making a probe that would allow him to visualize the expression of interesting genes. It was a gamble — no one knew if there were additional proteins required to turn the sequence into a glowing final product…but they discovered that they could get functional product in bacteria within a month.

They published a paper describing GFP as a new marker for gene expression, which Science disliked because of the simple title, and so they had to give it a cumbersome title for the reviewers, which got changed back for publication. They had a beautiful cover photo of a glowing neuron in the living animal.

Advantages of GFP: heritable, relatively non-invasive, small and monomeric, and visible in living tissues. Roger Tsien worked to improve the protein and produce variants that fluroesced at different wavelengths. There are currently at least 30,000 papers published that use fluroescent proteins, in all kinds of organisms, from bunnies to tobacco plants.

He showed some spectacular movies from Silverman-Gavrila of dividing cells with tubulin/GFP, and another of GFP/nuclear localization signal in which nuclei glowed as they condensed after division, and then disappeared during mitosis. Sanes and Lichtman’s brainbow work was shown. Also cute: he showed the opening sequence of the Hulk movie, which is illustrated with jellyfish fluorescence (he does not think the Hulk is a legitimate example of a human transgenic.)

Finally, he returned to his mechanoreceptor work and showed the transducing cells in the worm. One of the possibilities this opened up was visual screening for new mutants: either looking for missing or morphologically aberrant cells, or even more subtle things, like tagging expression of synaptic proteins so you can visually scan for changes in synaptic function or organization.

He had a number of questions he could address: how are mechanotransducers generated, how is touch transduced, what is the role of membrane lipids, can they identify other genes important in touch, and what turns off these genes?

They traced the genes involved in turning on the mec-3 gene; the pathway, it turned out, was also expressed in other cells, but they thought they identified other genes involved in selectively regulating touch sensitivity. One curious thing: the mec genes are transcribed in other cells that aren’t sensitive, but somehow are not translated.

They are searching for other touch genes. The touch screen misses some relevant genes because they have redundant alternatives, or are pleiotropic so other phenotypes (like lethality) obscure the effect. One technique is RNAi, and they made an interesting observation. Trying about 17000 RNAis, they discovered that 600 had interesting and specific effects, 1100 were lethal, and about 15,000 had no effect at all. The majority of genes are complete mysteries to us. They’ve developed some techniques to get selective incorporation of RNAis into just neurons of C. elegans, so they’re hoping to uncover more specific neural effects. One focus is on the integrin signaling pathway in the nervous system, which they’ve knocked out and found that it demolishes touch sensitivity — a new target!

They are now using a short-lived form of GFP that shuts down quickly, so they’ve got a sharper picture of temporal patterns of gene activity.

Chalfie’s summary:

  • Scientific progress is cumulative.

  • Students and post-docs are the lab innovators.

  • Basic research is essential. Who would have thought working on jellyfish would lead to such powerful tools?

  • All life should be studied; not just model organisms.

Chalfie is an excellent speaker and combined a lot of data with an engaging presentation.