PROFILE

Profile of espite having a bad reputa- tion, cholesterol is an essen- tial component of the plasma membranes of animal cells, whereD it is thought to modulate the properties of the lipid bilayer. Choles- terol can also bind directly to proteins in the membrane. In his Inaugural Article published in 2006, Martin Chalfie, the William R. Kenan, Jr., Professor of Bio- logical Sciences at (New York), in collaboration with Thomas Benzing (University of Cologne, Germany), identified a new class of cholesterol-binding proteins among the prohibitin (PHB)-domain protein family (1). PHB-domain proteins appear to regulate a variety of membrane func- tions, from cell signaling to mech- anosensation. Studying two members of the family, MEC-2 and Podocin, Chalfie, Benzing, and their colleagues found that cholesterol is crucial for the activity of two different classes of channel proteins to which the PHB-domain proteins bind. They suggest that the binding of choles- terol by the PHB-proteins alters the lo- cal lipid environment of associated membrane proteins and changes their activity. Questioning Research Chalfie, born in 1947 and elected to the National Academy of Sciences in 2004, grew up in Chicago. As a child, all ele- Martin Chalfie ments of science interested him, but he feels his early activities were somewhat Hastings provided Chalfie with one of Chalfie took a series of short-term jobs, mundane. ‘‘Unfortunately, I did not have including a stint selling dresses for his that real indicator of a career in science his fondest memories of Harvard. ‘‘I parents’ dress manufacturing business in that many of my friends have. I did not never seemed to be able to get to the Chicago. In 1970 he began teaching high make explosives and almost destroy my biology library when it was open, so I school at Hamden Hall Country Day home,’’ he says. As a child, he cut out asked Woody for permission to get a School (Hamden, CT). newspaper comics about for library key for late night reading,’’ he Chalfie took the advice of a fellow a scrapbook and, in high school, partici- recalls. ‘‘Most of my professors seemed teacher and applied to work in the labo- pated in a weekly science club after very distant, so I was amazed when he ratory of Jose Zadunaisky at Yale Uni- school. ‘‘I was fairly good at science in got up from his desk, walked down four versity (New Haven, CT) during the school,’’ he recalls. ‘‘That was the positive flights of stairs to the library office, and summer of 1971. During the initial in- reinforcement to keep me going.’’ Chalfie said, ‘Give this boy a key.’ I’ve since terview, Zadunaisky told Chalfie about entered Harvard University (Cambridge, learned that this kindness was character- MA) in 1965 and thought that he would istic of him.’’ his work measuring chloride transport in major in math. He soon switched gears. Chalfie spent the summer after his the frog cornea by using an Ussing ‘‘I was attracted to biochemistry because junior year working in the laboratory chamber. ‘‘I thought about the paper I I could do a little bit of everything: chem- of Klaus Weber at Harvard. Chalfie set had written for Woody Hastings, and istry, math, and biology,’’ he explains. out to study the active site of aspartate forgetting that that research involved ‘‘The subject also seemed new and transcarbamylase, but ‘‘although I kept sodium, not chloride, transport, toad exciting.’’ trying to do the experiments, I failed and not frog, and bladder and not During his junior year, Chalfie took miserably all summer,’’ Chalfie says. ‘‘I cornea—although it did measure trans- a cell physiology class with Woody Hast- decided I shouldn’t be in science.’’ So port with a Ussing chamber—I tried to ings, but Chalfie could not register for for his senior year, Chalfie took the last impress Jose by asking if cAMP was in- the laboratory portion and wrote a pa- required course for the biochemistry volved,’’ Chalfie recalls. ‘‘He liked the per instead. The subject, the role of cy- major and then other courses that inter- ested him, including law, theater, and clic AMP (cAMP) in sodium transport This is a Profile of a recently elected member of the National in the toad bladder, would later spark Russian literature. Academy of Sciences to accompany the member’s Inaugural an idea that led to Chalfie’s first pub- After graduating in 1969 and still un- Article on page 17079 in issue 46 of volume 103. lished research article. In the meantime, convinced about a career in research, © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704615105 PNAS ͉ February 5, 2008 ͉ vol. 105 ͉ no. 5 ͉ 1393–1395 Downloaded by guest on October 2, 2021 question but assigned me a completely was not sure what exactly to watch. At a mechanosensory current could have oc- different project. Jose then left to do loss, he decided to count the number of curred for many reasons, but the alter- research for the summer in France.’’ microtubules in each section. Instead of ation of the properties of the current Chalfie recalls, ‘‘Enamored with my own decreasing the further from the cell suggests that the channel proteins were idea and left on my own, I didn’t do the body (as would be expected if microtu- directly involved in transduction,’’ he assigned project but tried to see if in- bules started in the cell body), the count explains. Chalfie is not done with his creasing cAMP affected the current. ‘‘fluctuated all over the place.’’ Nichol studies of mechanosensation. ‘‘Now that Fortunately, it did so dramatically, and Thomson helped Chalfie acquire more we have good evidence that we have Jose was very supportive when he found sections, and Chalfie soon showed that, identified the transduction complex, the out what I had done at the end of the contrary to prevailing theory, the micro- biggest problem lies ahead: How does summer.’’ This research would garner tubules did not span the entire length of this complex translate touch into an Chalfie his first publication (2) and give the nerve process but started and electrical response?’’ him the confidence to consider a career stopped along the way (6). in biology. Chalfie relished his time at the labo- An Enjoyable Collaboration ratory. ‘‘My time as a postdoc made me For his Inaugural Article (1), Chalfie stud- Insensitive Worms the scientist I am today,’’ he says. ‘‘The ied MEC-2, a protein associated with the For his doctoral work, Chalfie entered complete freedom, terrific colleagues, transduction complex needed for touch Harvard University’s physiology depart- and extensive material support that were sensitivity. Chalfie’s laboratory had found ment in 1972, where he chose Robert characteristic of the LMB were very that MEC-2, which is associated with the Perlman, ‘‘who still remains a terrific inner leaflet of the plasma membrane, friend,’’ as his thesis advisor. ‘‘Bob was greatly increases the current from acti- very patient and willing to listen to all ‘‘My time as a postdoc vated touch channel proteins in frog of my unformed and misinformed ideas. oocytes (12, 15). MEC-2 is a PHB-domain He was also a clear and deep thinker. made me the scientist protein; other PHB-proteins also appear He was soft-spoken, but what he said to regulate membrane functions. was important and thoroughly consid- I am today.’’ Chalfie recalls that in 2004, he re- ered,’’ recalls Chalfie. ceived a call from Benzing, who was For his thesis, Chalfie used cell sus- studying a similar PHB-domain protein, pensions from rat pheochromocytomas stimulating. Everything was provided; so called Podocin, which is found in mam- (adrenal tumors) to look at the biosyn- much so that you had no excuse for not malian kidneys. Benzing suggested that thesis and release of catecholamines (3). being able to work except your own lim- they collaborate to study how these pro- teins regulate activity. Together, they Chalfie initially looked for postdoctoral itations.’’ After five years in England, positions in the same field for his gradu- found that MEC-2 and Podocin share Chalfie left the laboratory in 1982. He ate work but soon decided that he wanted three properties: Both proteins bind to joined the faculty of Columbia Univer- to do something different. A visit from target proteins in membranes, to them- sity in the department of biological sci- high school friend Bob Horvitz, who was selves, and to cholesterol. Chalfie ex- ences and continued to study C. elegans doing a postdoc with Sydney Brenner at plains that multimerization and the touch mutants. He found that some mu- the MRC Laboratory of Molecular Biol- binding to cholesterol were significant tants were insensitive to touch because ogy (Cambridge, U.K.), prompted Chalfie because these events could ‘‘change the to apply to Brenner’s laboratory. the cells were missing or incompletely lipid environment around target proteins.’’ In 1977, Chalfie traveled to England differentiated, a result that led to the The activity of the different channel to work with Brenner. Although intend- study of how cell type is determined (7– proteins that bind MEC-2 and Podocin ing to study neurotransmitters in Caeno- 9). Other touch-insensitive mutants have could thus be modified by the presence rhabditis elegans, Chalfie instead took up fully formed but nonfunctional cells. of these PHB-domain proteins (1). a project started by John Sulston, then a These mutants led to investigations into Chalfie calls his collaboration with staff scientist in the laboratory, on the the molecular basis of mechanosensory Benzing ‘‘one of the most enjoyable I genes involved in mechanosensation. transduction (e.g., refs. 10–14). have had with anyone.’’ The two con- Sulston had earlier found that some During the 1980s and 1990s, Chalfie tinue to work on questions raised in the C. elegans mutants are unable to re- and his laboratory studied the molecular Inaugural Article, such as whether these spond to touch (4). Working with biology of the touch genes. Convincing proteins mediate effects of steroids, Sulston, Chalfie began studying these evidence that some of these genes en- which are cholesterol derivatives, at mutants and the cells that sensed touch; coded components of a transduction membranes. Typically, steroids are together they identified the first collec- complex, however, did not arise until thought to act by influencing transcrip- tion of genes needed for touch sensitiv- 2005 (14). At that time, one of his grad- tion, but some of their rapid effects may ity (5). ‘‘These genes have been keeping uate students, Bob O’Hagan, working be independent of transcription. Benzing me busy ever since,’’ Chalfie says. One with Miriam Goodman, a former post- and Chalfie are still studying whether discovery, however, came unexpectedly. doc who had established her own labo- PHB-domain proteins mediate these ‘‘The first real skill that I used in my ratory at Stanford University (Palo nongenomic effects. Chalfie explains postdoc was my ability to count,’’ he Alto, CA), recorded electrical responses that anecdotal evidence, including the remembers. The touch-sensing cells from the touch-sensing cells of wild-type finding that defects in human Podocin were easily identified in electron micro- and mutant C. elegans. The loss of some result in steroid-resistant nephrotic dis- graph because their processes were filled touch genes abolished the electrical re- ease, hint that PHB-domain proteins with prominent microtubules. One day, sponse to touch, confirming that the indeed could regulate membrane func- Chalfie decided to study the cells in the genes were necessary for the touch re- tion by binding steroids. serial sections of the worm that were sponse. Even more exciting to Chalfie, being used to reconstruct the animal’s particular missense mutations in channel Glow Worms neural anatomy. He felt he should look genes altered the nature of the electrical In 1994, Chalfie published what he at ‘‘his cells’’ as part of his research but response (14). ‘‘The absence of the considers his most influential paper,

1394 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0704615105 Davis Downloaded by guest on October 2, 2021 which describes the first use of the now- completed the sequence and unsuccess- quently, they put GFP into C. elegans, ubiquitous green fluorescent protein fully tried to contact him. Not until and GFP’s life as a biomarker began. (GFP) as a reporter of gene expression 1992, while searching a publications da- Although the paper focused on GFP (16). Chalfie first heard about GFP in tabase, did Chalfie discover Prasher’s as a promoter-driven construct, it men- 1988 at a seminar given by Paul Brehm, publication of the GFP sequence (17). tioned that GFP could be conjugated to who now is at State University of New He got in touch with Prasher, and they a protein, and Hazelrigg, by then also York (SUNY)-Stony Brook. At the reestablished their collaboration. on the faculty at Columbia, soon showed that to be the case (16, 18). time, Chalfie’s laboratory used lacZ fu- Chalfie acquired the GFP cDNA and ‘‘These papers were the beginning of sions to localize gene activity, and he Ghia Euschirken, a graduate student, the development of an ever-increasing thought, ‘‘What an incredible tool this made a construct to express the pro- number of applications of GFP and would be, especially in C. elegans, which, tein in E. coli. When he started this other fluorescent proteins, most of we never tire of telling people, is trans- work, Chalfie recalls that researchers which I would never have imagined. I parent.’’ Chalfie found that Doug knew that the fluorescent protein am amazed and proud at what has hap- Prasher at Woods Hole Oceanographic formed from a modification of the pened with GFP.’’ Chalfie is also de- Institute (Woods Hole, MA) was at- polypeptide chain but had no added lighted with the directions his work has tempting to clone the GFP cDNA from fluorophore. taken over the years, such as studies of the jellyfish Aequorea victoria. The two At the time, he explains, they did not neurodegeneration and the development brainstormed about what they could do know how the modification was made; of combinatorial methods to label and with the full-length cDNA and agreed many thought that GFP might require kill cells. Still, he is quick to credit oth- to keep in touch. Chalfie then married one or more enzymes to affect the ers, ‘‘In large part, the lab’s success is Tulle Hazelrigg and joined her at the changes. Chalfie gambled on the possi- due to students, postdocs, and collabo- (Salt Lake City, UT) bility that GFP needed no other jellyfish rators who have pushed the work for a sabbatical in 1989. During that protein, and, ‘‘when Ghia’s coli glowed along.’’ time, unbeknownst to Chalfie, Prasher green, we knew we were right.’’ Subse- Tinsley Davis, Freelance Science Writer

1. Huber TB, et al. (2006) Podocin and MEC-2 bind cho- 7. Way JC, Chalfie M (1988) mec-3, a homeobox-contain- 13. Chelur DS, et al. (2002) The mechanosensory protein lesterol to regulate the activity of associated ion chan- ing gene that specifies differentiation of the touch MEC-6 is a subunit of the C. elegans touch-cell de- nels. Proc Natl Acad Sci USA 103:17079–17086. receptor neurons in C. elegans. Cell 54:5–16. generin channel. Nature 420:669–673. 2. Chalfie M, Neufeld AH, Zadunaisky JA (1972) Action of 8. Xue D, Tu Y, Chalfie M (1993) Cooperative interactions 14. O’Hagan R, Chalfie M, Goodman MB (2005) The MEC-4 epinephrine and other cyclic AMP-mediated agents on between the Caenorhabditis elegans homeoproteins DEG/ENaC channel of Caenorhabditis elegans touch the chloride transport of the frog cornea. Invest Oph- UNC-86 and MEC-3. Science 261:1324–1328. receptor neurons transduces mechanical signals. Nat thalmol 11:644–650. 9. Mitani S, Du H, Hall DH, Driscoll M, Chalfie M (1993) Neurosci 8:43–50. 3. Chalfie M, Perlman RL (1976) Studies of a transplant- Combinatorial control of touch receptor neuron ex- 15. Huang M, Gu G, Ferguson EL, Chalfie M (1995) A sto- able rat pheochromocytoma: Biochemical character- pression in Caenorhabditis elegans. Development matin-like protein necessary for mechanosensation in ization and catecholamine secretion. Pharmacol Exp 119:773–783. C. elegans. Nature 378:292–295. Ther 197:615–622. 10. Driscoll M, Chalfie M (1991) The mec-4 gene is a mem- 16. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC 4. Sulston J, Dew M, Brenner S (1975) Dopaminergic neu- ber of a family of Caenorhabditis elegans genes that (1994) Green fluorescent protein as a marker for gene rons in the nematode Caenorhabditis elegans. Comp can mutate to induce neuronal degeneration. Nature expression. Science 263:802–805. Neurol 163:215–226. 349:588–593. 17. Prasher DC, Eckenrode VK, Ward WW, Prendergast 5. Chalfie M, Sulston J (1981) Developmental genetics of 11. Huang M, Chalfie M (1994) Gene interactions affecting FG, Cormier MJ (1992) Primary structure of the Ae- the mechanosensory neurons of Caenorhabditis el- mechanosensory transduction in Caenorhabditis el- quorea victoria green-fluorescent protein. Gene egans. Dev Biol 82:358–370. egans. Nature 367:467–470. 111:229–233. 6. Chalfie M, Thomson JN (1979) Organization of neuro- 12. Goodman MB, et al. (2002) MEC-2 regulates C. elegans 18. Wang S, Hazelrigg T (1994) Implications for bcd mRNA nal microtubules in the nematode Caenorhabditis el- DEG/ENaC channels needed for mechanosensation. Na- localization from spatial distribution of exu protein in egans. J Cell Biol 82:278–289. ture 415:1039–1042. Drosophila oogenesis. Nature 369:400–403.

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