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Obituary Roger Y. Tsien (1952–2016)
BBAMCR-18050; No. of pages: 2; 4C: Biochimica et Biophysica Acta xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr
Obituary Roger Y. Tsien (1952–2016) On August 24, 2016, Roger Y. Tsien prematurely died at the age of 64, while riding his bicycle close to his new home in Eugene. The extraordinary scientific contributions of Roger, Nobel laureate in Chemistry, have been remembered in many articles in scientific journals, academia obituaries and newspapers. For me, Roger was also a scientific collaborator and mentor but, above all, he was a lifelong friend, one of the persons to whom I turned for suggestions and advice in difficult moments of my life. This short text will not be focused on his many revolutionary contributions to biomedicine but primarily on remembering a friend and our scientific and personal relationship. I met Roger about 40 years ago, in the winter of 1977, at Cambridge University. I remember well how he was dressed when we first met (he dressed like this most of his life): jeans, biking clips on his trousers, a sort of leather holster attached to his belt carrying a Hewlett-Packard calculator, and numerous coloured pens in his shirt pocket. I mention the calculator because I still remember the speed with which he clicked its buttons if you asked him to calculate the amounts of Ca2+ and EGTA necessary for making a Ca2+ solution buffered at some submicromolar concentration. Something that took everyone else many minutes (and often with wrong results) Roger would do in no time at all. He was a post doc with Tim Rink at the Department of Physiology and I was spending a few months at the Department of Biochemistry. I remember Tim, usually quite critical and cynical, openly saying “Roger is one of the most intelligent people I ever met in my life”. Initially, I thought he was exaggerating but he was perfectly right. At the time, Roger was bringing to perfection Ca2+-selective microelectrodes (that eventually led to the seminal Nature article with his brother Dick, Tim Rink and Eduardo Marban), measuring Ca2+ in resting and contracting heart myocytes. In his “spare time”, he was busy with his project on new Ca2+ indicators and also on membrane potential probes. I returned to Cambridge about a year later and that was when my friendship with Roger really started, in those long and dark afternoons of the English winter. Roger was not very talkative and people thought that he was arrogant; he was simply shy and reserved. The reason we became friends is inextricably linked to the English tradition of pubs opening at 6 p.m.: most of the fellows left around that time and went to the pub for a pint, but not Roger and I. Because of my Italian habits, I do not usually drink beer before dinner, while Roger had his Chinese variant of alcohol dehydrogenase (that makes people more sensitive to alcoholic beverages). Let it be known, however, that Roger did sometimes drink a bit of wine over dinner and, at times, also a good quality Scotch after dinner. In those days, Tim Rink was sharing a grant with my boss, Jim Metcalfe, and others. For this reason, Roger had been offered a bench in Biochemistry as it was better equipped for chemical synthesis than his lab in Physiology. So, alone in the lab after 6 p.m., I used to go over to Roger's bench – always full of tubes and little bottles with powders of many different colours – and chat about work and our personal lives. I vividly remember one of those afternoons coming back from the library (we used to go to the library in those days!), meeting Roger and mentioning a very interesting
paper that I had just read in Biochemistry by Thomas and Racker. It was about the use of an ester of fluorescein (fluorescein diacetate) that could be trapped in live cells and used to measure cytosolic pH. “Did you read it?”, I asked Roger. “Yes. Interesting.”, he answered. “Wouldn't it be nice to use this same method to trap one of your dyes?”, I pressed on. “Yes. I thought of it.”, was his simple reply after which we continued chatting about other stuff. A few months later, again after 6 p.m., I was struggling with a new flame photometry instrument, trying to measure the Ca2+ content of different samples. Roger came over to me and, in his typical harsh way, said “Why are you wasting your time with those useless experiments?… Come with me to Tim's lab with your lymphocytes and let's try one of my dyes…”. I took my cells and we added a few microliters of a yellowish solution (it was quin2/AM) to an aliquot of the lymphocytes. After half an hour we put them in a cuvette and measured the excitation spectrum. “It is the right one, the AM ester has been hydrolyzed”, Roger said “Let's add a Ca2+ ionophore” (it was A23187). And there, right in front of our eyes, we saw the fluorescence going up very rapidly! I was thrilled and so were Roger and Tim. We had a method to measure Ca2+ in a small live cell, and it worked the first time! I immediately phoned my wife saying that I was going to come home late that evening and we went on to do the usual and obvious controls. The method really worked and, as the saying goes, the rest is history; AM esters of Ca2+ dyes had an enormous success in the scientific community and they are still (36 years later) extensively used in thousands of laboratories around the world. A couple of months later I had to return to Italy and during those hectic days we exchanged information through long letters. International telephone calls were too expensive at the time (and, believe it or not, email did not yet exist). We shared our enthusiasm and frustration for the experiments carried out in Cambridge (primarily on platelets) and in Padua (on lymphocytes) that eventually led to the popular papers of 1982 in Nature and J Cell Biol. I was back in Cambridge a few times during that period and our friendship continued to grow. I met Wendy, his girlfriend at that time, he also came to Padua, and we published a few good papers together before Roger accepted the offer from Berkeley and decided to cross the Atlantic. Roger loved Cambridge in particular and England in general and I am pretty sure that if he had been made a good offer he would have stayed there. But it didn't happen. Once in Berkeley, he invited me to his lab. Together with my wife and our 3-year-old daughter, Lucia, we spent three months in California in the summer of 1984. There, I worked on a new membrane-permeable heavy metal chelator (TPEN, rediscovered and resynthesized by Roger) that allowed us to understand the heavy metal problem that plagued some experiments with quin2. Most of the time, though, I spent testing new Ca2 + indicators that Greg Grinkiewicz was synthetizing. Among these, one appeared quite promising; it was called fura-2… Eventually that paper was published and the lives of most of us changed forever. Ca2+ dyes were not the only molecules that Roger invented in those years. Just to mention the most popular, he was responsible for the pH indicator BCECF, Na+ and K+ dyes, new caged Ca2+ chelators, caged IP3, and membrane potential dyes. The extraordinary and unique quality of Roger was that of identifying an important biological problem and then going on to invent a molecule to address it.
http://dx.doi.org/10.1016/j.bbamcr.2017.02.008 0167-4889/
Please cite this article as: B.T. Pozzan, Obituary Roger Y. Tsien (1952–2016), Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/ j.bbamcr.2017.02.008
2
B.T. Pozzan / Biochimica et Biophysica Acta xxx (2017) xxx–xxx
My collaboration with Roger continued over the years and we visited each other every now and then. On one of those occasions we were in the Dolomites and went skiing together. He enjoyed skiing but, after so many years, there was finally one thing that I could do better than Roger! At the end of 1989, just before he moved to San Diego, I was again in Berkeley. One evening, his wife Wendy was out and we were alone for dinner. I have always loved Chinese food but Roger hated the standard stuff found in most Chinese restaurants. Perhaps unsurprisingly, he remembered the recipes that his mother used to prepare when he was a child and so it was that I discovered one of Roger's hidden talents. I am probably one of the few people who have tasted food made by Roger with his own hands – and it was delicious! After dinner, as we were walking through the Berkeley campus, I told him about our idea of trying to use recombinant aequorin to measure Ca2+ in organelles… He said it was a good idea but his mind was on something different, using another protein made by Aequorea victoria. He was alluding to GFP, of course, a protein unknown to most of us in those days. He started talking about the generation of a chromophore by cyclization of three amino acids and a lot of other details that I hardly understood. I heard nothing else about this mysterious protein for the next five years, until the famous Science paper by Chalfie and Prasher came out in 1994. Roger had been scooped mainly because, in those days, his lab had little experience in molecular biology (Roger Heim and Atsushi Miyawaki had just joined his group). He was quick to take further and substantial steps in the field: the generation of differently coloured GFPs and the demonstration of the possibility of obtaining FRET sensors based on blue- and green-mutants of GFP. Together, we published the first paper on the expression of GFP in mammalian cells and within an organelle (mitochondria) of a living cell. The story of his contribution to the GFP saga over the last 20 years is widely known: from the crystallization of GFP to the generation of monomeric RFPs, passing through the revolutionary method for generating GFP mutants using the B lymphocyte mechanism for producing different antibodies, from the GFP-based sensors for Ca2+ to those for cAMP, cGMP and protein phosphorylation. Roger was a good pianist and playing the piano was his main hobby. I heard him playing a number of times but I will never forget two of them. The first was in Cambridge, a long time ago, at the time of the first quin-2 experiments. I had been invited for dinner by A.L. Lew, an expert on red blood cell Ca2+ who was working at the Physiology Department, close to Roger's lab. Roger played the piano, Lew the violin and together they played a Mozart piece. I thought it was fantastic but Roger, always the perfectionist, was very critical of his performance. The second episode is much more recent, after he had the stroke in 2013. I was visiting him in San Diego, in the new house. Early one morning I heard Roger playing the piano. He was trying to reactivate the function of his right hand, affected by the stroke. He was playing simple scales on the piano, with noticeable difficulty. He paused, looked at me and I saw tears in his eyes. What is perhaps less known to the majority of people is Roger's unbridled generosity, both on the scientific and personal levels. Two simple examples are enough to illustrate this lesser known facet. The first was recounted to me by his wife several years ago. At the time Roger had founded Aurora Bioscience and I was talking to Wendy about his interest in starting up a company. “Roger is not interested in making money,” she told me “we have more than enough for our needs. He created this company mainly because it is a way to find a job for his fellows that could not make it into academia”. The second story concerns Doug Prasher. Doug is the person who rediscovered GFP, cloned it and had a major contribution in the famous 1994 paper of Martin Chalfie in C. elegans. In a series of unfortunate events, Prasher was not recognized for his fundamental discoveries, eventually lost his job and, by the time the Nobel Prize was awarded for GFP, he was working as a school bus driver. Nobel laureates have the privilege of inviting a very small and limited number of guests to the award ceremony and Roger invited Doug Prasher to Stockholm. Not only that but, shortly afterwards, he offered Doug a position in his lab, where he is still working. Many scientists around the world have examples of Roger's generosity in providing suggestions, new compounds and fresh ideas. We are all
indebted to him. We all will miss him, for his creativity in science and for his profound human qualities. Ciao, Roger!
By Tullio Pozzan Available online xxxx
Please cite this article as: B.T. Pozzan, Obituary Roger Y. Tsien (1952–2016), Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/ j.bbamcr.2017.02.008
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr
Obituary Roger Y. Tsien (1952–2016) On August 24, 2016, Roger Y. Tsien prematurely died at the age of 64, while riding his bicycle close to his new home in Eugene. The extraordinary scientific contributions of Roger, Nobel laureate in Chemistry, have been remembered in many articles in scientific journals, academia obituaries and newspapers. For me, Roger was also a scientific collaborator and mentor but, above all, he was a lifelong friend, one of the persons to whom I turned for suggestions and advice in difficult moments of my life. This short text will not be focused on his many revolutionary contributions to biomedicine but primarily on remembering a friend and our scientific and personal relationship. I met Roger about 40 years ago, in the winter of 1977, at Cambridge University. I remember well how he was dressed when we first met (he dressed like this most of his life): jeans, biking clips on his trousers, a sort of leather holster attached to his belt carrying a Hewlett-Packard calculator, and numerous coloured pens in his shirt pocket. I mention the calculator because I still remember the speed with which he clicked its buttons if you asked him to calculate the amounts of Ca2+ and EGTA necessary for making a Ca2+ solution buffered at some submicromolar concentration. Something that took everyone else many minutes (and often with wrong results) Roger would do in no time at all. He was a post doc with Tim Rink at the Department of Physiology and I was spending a few months at the Department of Biochemistry. I remember Tim, usually quite critical and cynical, openly saying “Roger is one of the most intelligent people I ever met in my life”. Initially, I thought he was exaggerating but he was perfectly right. At the time, Roger was bringing to perfection Ca2+-selective microelectrodes (that eventually led to the seminal Nature article with his brother Dick, Tim Rink and Eduardo Marban), measuring Ca2+ in resting and contracting heart myocytes. In his “spare time”, he was busy with his project on new Ca2+ indicators and also on membrane potential probes. I returned to Cambridge about a year later and that was when my friendship with Roger really started, in those long and dark afternoons of the English winter. Roger was not very talkative and people thought that he was arrogant; he was simply shy and reserved. The reason we became friends is inextricably linked to the English tradition of pubs opening at 6 p.m.: most of the fellows left around that time and went to the pub for a pint, but not Roger and I. Because of my Italian habits, I do not usually drink beer before dinner, while Roger had his Chinese variant of alcohol dehydrogenase (that makes people more sensitive to alcoholic beverages). Let it be known, however, that Roger did sometimes drink a bit of wine over dinner and, at times, also a good quality Scotch after dinner. In those days, Tim Rink was sharing a grant with my boss, Jim Metcalfe, and others. For this reason, Roger had been offered a bench in Biochemistry as it was better equipped for chemical synthesis than his lab in Physiology. So, alone in the lab after 6 p.m., I used to go over to Roger's bench – always full of tubes and little bottles with powders of many different colours – and chat about work and our personal lives. I vividly remember one of those afternoons coming back from the library (we used to go to the library in those days!), meeting Roger and mentioning a very interesting
paper that I had just read in Biochemistry by Thomas and Racker. It was about the use of an ester of fluorescein (fluorescein diacetate) that could be trapped in live cells and used to measure cytosolic pH. “Did you read it?”, I asked Roger. “Yes. Interesting.”, he answered. “Wouldn't it be nice to use this same method to trap one of your dyes?”, I pressed on. “Yes. I thought of it.”, was his simple reply after which we continued chatting about other stuff. A few months later, again after 6 p.m., I was struggling with a new flame photometry instrument, trying to measure the Ca2+ content of different samples. Roger came over to me and, in his typical harsh way, said “Why are you wasting your time with those useless experiments?… Come with me to Tim's lab with your lymphocytes and let's try one of my dyes…”. I took my cells and we added a few microliters of a yellowish solution (it was quin2/AM) to an aliquot of the lymphocytes. After half an hour we put them in a cuvette and measured the excitation spectrum. “It is the right one, the AM ester has been hydrolyzed”, Roger said “Let's add a Ca2+ ionophore” (it was A23187). And there, right in front of our eyes, we saw the fluorescence going up very rapidly! I was thrilled and so were Roger and Tim. We had a method to measure Ca2+ in a small live cell, and it worked the first time! I immediately phoned my wife saying that I was going to come home late that evening and we went on to do the usual and obvious controls. The method really worked and, as the saying goes, the rest is history; AM esters of Ca2+ dyes had an enormous success in the scientific community and they are still (36 years later) extensively used in thousands of laboratories around the world. A couple of months later I had to return to Italy and during those hectic days we exchanged information through long letters. International telephone calls were too expensive at the time (and, believe it or not, email did not yet exist). We shared our enthusiasm and frustration for the experiments carried out in Cambridge (primarily on platelets) and in Padua (on lymphocytes) that eventually led to the popular papers of 1982 in Nature and J Cell Biol. I was back in Cambridge a few times during that period and our friendship continued to grow. I met Wendy, his girlfriend at that time, he also came to Padua, and we published a few good papers together before Roger accepted the offer from Berkeley and decided to cross the Atlantic. Roger loved Cambridge in particular and England in general and I am pretty sure that if he had been made a good offer he would have stayed there. But it didn't happen. Once in Berkeley, he invited me to his lab. Together with my wife and our 3-year-old daughter, Lucia, we spent three months in California in the summer of 1984. There, I worked on a new membrane-permeable heavy metal chelator (TPEN, rediscovered and resynthesized by Roger) that allowed us to understand the heavy metal problem that plagued some experiments with quin2. Most of the time, though, I spent testing new Ca2 + indicators that Greg Grinkiewicz was synthetizing. Among these, one appeared quite promising; it was called fura-2… Eventually that paper was published and the lives of most of us changed forever. Ca2+ dyes were not the only molecules that Roger invented in those years. Just to mention the most popular, he was responsible for the pH indicator BCECF, Na+ and K+ dyes, new caged Ca2+ chelators, caged IP3, and membrane potential dyes. The extraordinary and unique quality of Roger was that of identifying an important biological problem and then going on to invent a molecule to address it.
http://dx.doi.org/10.1016/j.bbamcr.2017.02.008 0167-4889/
Please cite this article as: B.T. Pozzan, Obituary Roger Y. Tsien (1952–2016), Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/ j.bbamcr.2017.02.008
2
B.T. Pozzan / Biochimica et Biophysica Acta xxx (2017) xxx–xxx
My collaboration with Roger continued over the years and we visited each other every now and then. On one of those occasions we were in the Dolomites and went skiing together. He enjoyed skiing but, after so many years, there was finally one thing that I could do better than Roger! At the end of 1989, just before he moved to San Diego, I was again in Berkeley. One evening, his wife Wendy was out and we were alone for dinner. I have always loved Chinese food but Roger hated the standard stuff found in most Chinese restaurants. Perhaps unsurprisingly, he remembered the recipes that his mother used to prepare when he was a child and so it was that I discovered one of Roger's hidden talents. I am probably one of the few people who have tasted food made by Roger with his own hands – and it was delicious! After dinner, as we were walking through the Berkeley campus, I told him about our idea of trying to use recombinant aequorin to measure Ca2+ in organelles… He said it was a good idea but his mind was on something different, using another protein made by Aequorea victoria. He was alluding to GFP, of course, a protein unknown to most of us in those days. He started talking about the generation of a chromophore by cyclization of three amino acids and a lot of other details that I hardly understood. I heard nothing else about this mysterious protein for the next five years, until the famous Science paper by Chalfie and Prasher came out in 1994. Roger had been scooped mainly because, in those days, his lab had little experience in molecular biology (Roger Heim and Atsushi Miyawaki had just joined his group). He was quick to take further and substantial steps in the field: the generation of differently coloured GFPs and the demonstration of the possibility of obtaining FRET sensors based on blue- and green-mutants of GFP. Together, we published the first paper on the expression of GFP in mammalian cells and within an organelle (mitochondria) of a living cell. The story of his contribution to the GFP saga over the last 20 years is widely known: from the crystallization of GFP to the generation of monomeric RFPs, passing through the revolutionary method for generating GFP mutants using the B lymphocyte mechanism for producing different antibodies, from the GFP-based sensors for Ca2+ to those for cAMP, cGMP and protein phosphorylation. Roger was a good pianist and playing the piano was his main hobby. I heard him playing a number of times but I will never forget two of them. The first was in Cambridge, a long time ago, at the time of the first quin-2 experiments. I had been invited for dinner by A.L. Lew, an expert on red blood cell Ca2+ who was working at the Physiology Department, close to Roger's lab. Roger played the piano, Lew the violin and together they played a Mozart piece. I thought it was fantastic but Roger, always the perfectionist, was very critical of his performance. The second episode is much more recent, after he had the stroke in 2013. I was visiting him in San Diego, in the new house. Early one morning I heard Roger playing the piano. He was trying to reactivate the function of his right hand, affected by the stroke. He was playing simple scales on the piano, with noticeable difficulty. He paused, looked at me and I saw tears in his eyes. What is perhaps less known to the majority of people is Roger's unbridled generosity, both on the scientific and personal levels. Two simple examples are enough to illustrate this lesser known facet. The first was recounted to me by his wife several years ago. At the time Roger had founded Aurora Bioscience and I was talking to Wendy about his interest in starting up a company. “Roger is not interested in making money,” she told me “we have more than enough for our needs. He created this company mainly because it is a way to find a job for his fellows that could not make it into academia”. The second story concerns Doug Prasher. Doug is the person who rediscovered GFP, cloned it and had a major contribution in the famous 1994 paper of Martin Chalfie in C. elegans. In a series of unfortunate events, Prasher was not recognized for his fundamental discoveries, eventually lost his job and, by the time the Nobel Prize was awarded for GFP, he was working as a school bus driver. Nobel laureates have the privilege of inviting a very small and limited number of guests to the award ceremony and Roger invited Doug Prasher to Stockholm. Not only that but, shortly afterwards, he offered Doug a position in his lab, where he is still working. Many scientists around the world have examples of Roger's generosity in providing suggestions, new compounds and fresh ideas. We are all
indebted to him. We all will miss him, for his creativity in science and for his profound human qualities. Ciao, Roger!
By Tullio Pozzan Available online xxxx
Please cite this article as: B.T. Pozzan, Obituary Roger Y. Tsien (1952–2016), Biochim. Biophys. Acta (2017), http://dx.doi.org/10.1016/ j.bbamcr.2017.02.008