Ras Then Rho and Rac

TURNING POINTS

GTPase switch: Ras then Rho and Rac

Anne Ridley

As a graduate student at Imperial Cancer just been identified as a GTPase-activating including serum, induced actin stress fibre Research Fund in London in the late 1980s, protein for Ras. This seemed to be an excel- assembly in serum-starved fibroblasts, simi- I worked on ‘oncogene cooperation’. My lent link to my graduate Ras and Schwann cell larly to the cellular response to microinjected graduate advisor, Hartmut Land, had shown days, as neurofibromatosis is a Schwann cell Rho. Alan Hall, being a chemist by training, that two different types of oncogenes, such cancer. I had even been to a neurofibromato- suggested I purify the factor in serum that as Ras and Myc, were needed to transform sis workshop where I had presented my data induced stress fibres. I really benefited from primary rodent fibroblasts into tumour-like on Ras in Schwann cells. My ideas changed, the lab expertise in purifying proteins using fibroblasts. I made the intriguing observation however, after a few trips to the library. Alan fast protein liquid chromatography (FPLC) in that expression of oncogenic Ras by itself in and Hugh had just published a paper showing the cold room — especially the tip to remem- primary Schwann cells inhibited proliferation, that a recently identified relative of Ras called ber to wear a thick sweater. We found that the whereas Ras in combination with Myc or SV40 RhoA induced dramatic changes to cell shape. mystery ‘factor’ in serum was bound to serum large T induced transformation. This finding After reading papers such as Dafna Bar-Sagi’s albumin, and was heat-insensitive but sensi- led me to collaborate with Hugh Paterson from report that injection of oncogenic Ras stimu- tive to various phospholipases. This allowed Chris Marshall’s laboratory in the Institute for lated membrane ruffling, and studies describ- us to identify the culprit as lysophosphatidic Cancer Research. Hugh had mastered the fine ing the effects of growth factors on the actin acid. The most exciting result came from art of microinjecting purified proteins into cytoskeleton, I became convinced that the link experiments with C3 transferase, a clostridial cells, and when he injected my Schwann cells between the actin cytoskeleton and Ras and enzyme that Klaus Aktories and Alan Hall had with oncogenic Ras protein, we found that Ras Rho proteins was the area I wanted to pursue, shown biochemically could modify RhoA, alone inhibited DNA synthesis. The power of and so I asked Hugh to teach me how to micro- and was therefore a great tool to inhibit RhoA microinjection as a tool to study immedi- inject cells. function. I showed that C3 transferase inhib- ate cellular responses to proteins struck me This was a major turning point that shaped ited the increase in stress fibres induced by at the time, but after I completed my PhD I my scientific career. The combination of cell serum and other stimuli, uncovering a link wanted to work in the US, and so moved to microinjection with Rho and Rac proteins between extracellular cues, RhoA activity and Boston armed with an EMBO postdoctoral together with stimulation with different types actin regulation. This detective work was great fellowship. However, I soon realised that the of growth factors, lipids and cytokines led us to fun, and highlights the excitement of being a research I was doing was not suited to me. identify the roles of Rho and Rac in regulating scientist that still keeps me going whenever When I discovered that Alan Hall, who worked the actin cytoskeleton. With microinjection, experiments are not going well. with Chris Marshall on Ras, was advertising a a whole experiment could be done and ana- The combination of cell biology with bio- postdoctoral position, I went for an interview lysed in a day: I would microinject proteins at chemistry was very important for the success and ended up taking the job and returning to multiple concentrations in the morning, and of this project, but the buzz in the Marshall and London. in some cases would stimulate the cells with Hall labs as other important discoveries (such Alan Hall’s and Chris Marshall’s offices and growth factors or cytokines. After fixing and as the link from Ras to MAPK) were being labs were right next to each other, sharing staining the cells, I would take reels of pho- made, and the freely shared expertise and equipment and Hugh’s expertise, and provid- tos. Finally, I would develop the films by hand reagents, also contributed a lot. In the twenty ing a fantastic environment in which to work and make the prints myself in the dark room. years since this work was performed, we as a postdoctoral fellow. I considered initially This sounds very old-fashioned now that we have learnt much more about Rho GTPases, working on neurofibromatosis 1, which had have digital images, but it provided me with but many unanswered questions remain. complete control over the whole process from Identifying the as-yet unknown actions of Anne Ridley is in the Randall Division of Cell and culturing the cells to visualizing them on pho- many of the twenty human Rho family mem- Molecular Biophysics, King’s College London, New tographic paper. bers, as well as of their numerous relatives in Hunt’s House, Guy’s Campus, London, SE1 1UL, UK. Using these approaches, I first showed other eukaryotes, will open exciting research e-mail: [email protected] that several different extracellular stimuli, paths in the coming years.

NATURE CELL BIOLOGY VOLUME 15 | NUMBER 4 | APRIL 2013 337