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An interview with Randy Schekman and Thomas Südhof
Scientific Life: TrendsTalk
Special Issue: Membrane Trafficking
An interview with Randy Schekman and Thomas Su¨dhof
In 2013 the Nobel Prize in Physiology or Medicine was awarded to Randy Schekman, James Rothman, and Thomas Su¨dhof for elucidating the molecular mechanisms behind membrane trafficking. Some molecules are too large to diffuse through membranes within the cell; therefore, the cell packages these molecules into small vesicles that deliver the cargo to destinations within or outside the cell. These trafficking mechanisms are crucial for proper hormone secretion, neurotransmission, and metabolic regulation. We are delighted that Randy Schekman (left) and Thomas Su¨dhof (right) are able to contribute their perspective on the field and discuss their reaction to receiving science’s highest honor. Randy Schekman is an Investigator of the Howard Hughes Medical Institute and a Professor in the Department of Molecular and Cell Biology at the University of California, Berkeley. Schekman received his BA from the University of California, Los Angeles (UCLA) and his PhD in Biochemistry from Stanford University. He completed his thesis work in the laboratory of Dr Arthur Kornberg, who won the Nobel Prize for identifying DNA polymerase. Schekman and his laboratory study the molecular mechanisms behind membrane assembly and vesicular traffic. A major focus of the laboratory is the secretory pathway – proteins destined for secretion are sorted into vesicles at the endoplasmic reticulum (ER), which proceed to the Golgi and later fuse with the plasma membrane where the proteins are released to the outside of the cell. Through a combination of genetic and cytologic approaches in yeast, work in Schekman’s laboratory led to the identification of a set of genes responsible for the events of the early secretory pathway. In addition, his laboratory has characterized the Sec61 translocation complex, the COPII vesicle coat complex, and inter-organelle transport vesicles. His laboratory is now applying the mechanisms discovered in yeast to studies of genetic diseases of protein transport such as rare craniofacial disorders and familial forms of Alzheimer’s disease. Thomas Su¨dhof is an Investigator of the Howard Hughes Medical Institute and Avram Goldstein Professor of Molecular and Cellular Physiology at Stanford University. Su¨dhof obtained his MD from the University of Go¨ttingen Medical School in Germany and completed his doctoral work in the laboratory of Dr Victor P. Whittaker at the Max Planck Institute for Biophysical Chemistry. Su¨dhof and his laboratory are interested in understanding how synapses form, how their properties are specified, and how they achieve precise signaling. 6
Failure to form synapses underlies disorders such as autism and schizophrenia, whereas improper neurotransmission is a key driver of neurodegenerative diseases. Two major focuses of the laboratory are synaptic cell adhesion molecules involved in synapse formation and the mechanisms regulating nerve terminal release of neurotransmitters. Through a combination of biophysical and biochemical techniques, work in Su¨dhof’s laboratory has led to the identification of several proteins that regulate SNARE-mediated neurotransmitter release, including synaptotagmin, complexin, and Munc18-1. In addition, his team has identified two trans-synaptic cell adhesion molecules, neurexins and neuroligins, which are crucial for proper synaptic function. He is currently focusing on understanding how these pre- and postsynaptic proteins control synapse formation and function during higher brain functions and how they can become impaired in neuropsychiatric diseases. Did you always want to be a researcher? Randy Schekman: My interest in science blossomed in junior high school with the annual science fair project. It deepened in college when I was introduced to experimental work in my freshman year at UCLA. At that point I lost interest in going to medical school and devoted myself to the lab, with decreasing attention to my coursework! I was greatly influence by Watson’s first edition of The Molecular Biology of the Gene and his autobiographical book, The Double Helix. The realization that one could use creativity, passion and hard work to probe the workings of the cell set me on the path to an academic research career. Thomas Su¨dhof: No – I initially wanted to be a practicing physician. It wasn’t until I performed research that I realized how much I loved it! How did you decide to focus on membrane assembly and vesicle trafficking? RS: I was influenced by my graduate advisor Arthur Kornberg, but even more by my best friend Bill Wickner who joined Kornberg’s lab fresh from graduate research in Eugene Kennedy’s lab at Harvard Medical School. My interests grew as a postdoctoral fellow in S.J. Singer’s lab at UCSD where I learned what was known about membrane traffic and where I first read George Palade’s brilliant work on the organization of the secretory pathway from his Nobel Lecture, which was published in Science the year he won the Nobel Prize. When I moved to UC Berkeley I resolved to work on this process in S. cerevisiae, motivated largely by the success of Lee Hartwell in using yeast Trends in Cell Biology, January 2014, Vol. 24, No. 1
Scientific Life: TrendsTalk genetics to dissect the cell division cycle. I was convinced that a combined genetic and biochemical approach to the secretory pathway in yeast would yield molecular mechanistic information that was not accessible using the then traditional tools of the morphological cell biologist. TS: All brain function depends on synaptic transmission. Neurotransmitter release initiates all synaptic transmission – it is half of the overall process, the other half being the sensing of the released neurotransmitters by postsynaptic receptors. I became interested in neurotransmitter release, which represents a vesicular trafficking event, because of its fundamental importance in brain function. I also became fascinated by it because of its unusual properties: it is extremely fast, occurring in a few hundred microseconds, tightly regulated by calcium, and highly plastic. What was your reaction when you received the call from Stockholm? RS: I was groggy from having just returned from Frankfurt the night before. Although I was aware that the Nobel decision would be announced that night, I had more or less given up on the idea that my work would be recognized by them. Therefore, the call really caught me by surprise, although by the time I stumbled over to the phone I was pretty sure who was calling. The feeling was surreal and it took a few moments to realize this was actually happening. TS: I was driving in the middle of Spain, somewhat lost, and my first thought was, ‘My God this is not true’ and then, ‘My God, this is wonderful.’ When you made your discovery, did you realize that it would have such a significant impact on the field? RS: When Peter Novick, one of my first graduate students, isolated our first secretion mutant – sec1 – I certainly had a strong feeling that this would allow us to develop molecular probes of the pathway and certainly lead to mechanistic insights. Nevertheless, I had no idea at the time that what we learned would have such direct relevance to the pathway in all eukaryotes, or that this knowledge could be applied in biotech companies to engineer the expression and secretion of important human proteins such as insulin. TS: My work consists not of a single discovery but of a series of observations that together explain different aspects of neurotransmitter release. There was no single paper or moment, but a series of revelations. Among them was synaptotagmin. The cloning already spawned the hypothesis that synaptotagmin is the calcium sensor for release (Perin et al., Nature 1990), and this was later supported by a series of biochemical experiments (Brose et al., Science 1992; Davletov and Sudhof, J. Biol. Chem. 1993) and studies on knockout mice (Geppert et al., 1994). The proof that synaptotagmin is the calcium sensor for release culminated with the demonstration that changing the calcium affinity of synaptotagmin changes the calcium affinity of release (Fernandez-Chacon et al., Nature 2001). A similar situation arose for the very important but overlooked question of how calcium channels localize next to release sites. When we cloned RIM we already knew that it would be important (Wang et al., Nature 1997), but it wasn’t until a series of knockout studies were performed that revealed how important RIM is for organizing neurotransmitter release (Schoch et al., Nature
Trends in Cell Biology January 2014, Vol. 24, No. 1
2002; Castillo et al., Nature 2002). This discovery culminated with the direct demonstration that RIM actually recruits calcium channels to the active zone, which was, in my view, an incredibly important observation (Kaeser et al., 2011). What do you think are the major outstanding questions in the field? RS: Many fascinating questions remain at the molecular mechanistic level about how vesicles form and how they are directed to their target and fuse with a target membrane. There is certainly enough to keep me interested and busy for the rest of my career. In my lab we are now working on the mechanism of autophagic membrane assembly and growth with a new cell-free system that recapitulates the first step in the biogenesis of the autophagosome. We are also keen to understand how large and unusually shaped cargo molecules such as collagen and lipoproteins are packaged into COPII transport vesicles that bud from the ER, how certain peroxisomal membrane proteins are packaged into transport vesicles that bud from the ER, how proteins destined for different cell surface domains are sorted at the trans-Golgi membrane, and how exosomal vesicles are generated at the cell surface or in multivesicular bodies. TS: Many questions remain open, some of which are detailed in a review I published in Neuron in November 2013. Fundamentally, we do not really understand the biophysics of fusion, we do not know which Sec1/Munc18like proteins are as essential for fusion as SNAREs are, and we still do not completely understand the mechanisms of presynaptic plasticity. There is so much to do! What is the best scientific advice that you have ever been given? RS: Focus. Perhaps this would have been my natural inclination, but what I learned from Kornberg was to focus on an important problem and remain dedicated to its solution. His specific advice was to pick an enzyme and study it. I didn’t follow that advice, but by his example I chose to study a pathway and, once the genetic approach showed promise, I reoriented my group to focus on that process using the genetic logic of Hartwell and the biochemical/enzymologic method I learned from Kornberg. TS: The best advice was not to worry about the journals I publish in, and not to worry about prizes. You have received the highest honor in the scientific community, what’s next? RS: Public education, and particularly public higher education, which was what made it possible for me to find my way in science. I am quite concerned that the next generation of aspiring young scholars from middle-class families may not have access to the great institutions that were available to me as I grew up. When I attended UCLA, beginning in 1966, I could pay the fees and room and board with income from a summer job. That is no longer possible, and families and students must go deep into debt to afford the same education that was given to me virtually free by the people of California. I intend to use whatever influence I have to make the case for continued investment in the University of California and in our other great public research universities. 7
Scientific Life: TrendsTalk TS: I really enjoy doing science and working with people, especially young people. Currently, most of my lab works on a completely unsolved problem related to autism and schizophrenia: how synapses are formed, specified, maintained, and restructured throughout life. I look forward to continuing this work. Finally, my hope is to contribute a little bit to the community, to the culture of science and university training, especially for the next generations.
8
Trends in Cell Biology January 2014, Vol. 24, No. 1
Figure acknowledgements: Randy Schekman and Thomas Su¨dhof, who with James Rothman shared the 2013 Nobel Prize for Physiology and Medicine. Photograph of R.S. (left) by H. Goren ß Howard Hughes Medical Institute; photograph of T.S. (right) ß S. Fisch. 0962-8924/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.11.006 Trends in Cell Biology, January 2014, Vol. 24, No. 1
Special Issue: Membrane Trafficking
An interview with Randy Schekman and Thomas Su¨dhof
In 2013 the Nobel Prize in Physiology or Medicine was awarded to Randy Schekman, James Rothman, and Thomas Su¨dhof for elucidating the molecular mechanisms behind membrane trafficking. Some molecules are too large to diffuse through membranes within the cell; therefore, the cell packages these molecules into small vesicles that deliver the cargo to destinations within or outside the cell. These trafficking mechanisms are crucial for proper hormone secretion, neurotransmission, and metabolic regulation. We are delighted that Randy Schekman (left) and Thomas Su¨dhof (right) are able to contribute their perspective on the field and discuss their reaction to receiving science’s highest honor. Randy Schekman is an Investigator of the Howard Hughes Medical Institute and a Professor in the Department of Molecular and Cell Biology at the University of California, Berkeley. Schekman received his BA from the University of California, Los Angeles (UCLA) and his PhD in Biochemistry from Stanford University. He completed his thesis work in the laboratory of Dr Arthur Kornberg, who won the Nobel Prize for identifying DNA polymerase. Schekman and his laboratory study the molecular mechanisms behind membrane assembly and vesicular traffic. A major focus of the laboratory is the secretory pathway – proteins destined for secretion are sorted into vesicles at the endoplasmic reticulum (ER), which proceed to the Golgi and later fuse with the plasma membrane where the proteins are released to the outside of the cell. Through a combination of genetic and cytologic approaches in yeast, work in Schekman’s laboratory led to the identification of a set of genes responsible for the events of the early secretory pathway. In addition, his laboratory has characterized the Sec61 translocation complex, the COPII vesicle coat complex, and inter-organelle transport vesicles. His laboratory is now applying the mechanisms discovered in yeast to studies of genetic diseases of protein transport such as rare craniofacial disorders and familial forms of Alzheimer’s disease. Thomas Su¨dhof is an Investigator of the Howard Hughes Medical Institute and Avram Goldstein Professor of Molecular and Cellular Physiology at Stanford University. Su¨dhof obtained his MD from the University of Go¨ttingen Medical School in Germany and completed his doctoral work in the laboratory of Dr Victor P. Whittaker at the Max Planck Institute for Biophysical Chemistry. Su¨dhof and his laboratory are interested in understanding how synapses form, how their properties are specified, and how they achieve precise signaling. 6
Failure to form synapses underlies disorders such as autism and schizophrenia, whereas improper neurotransmission is a key driver of neurodegenerative diseases. Two major focuses of the laboratory are synaptic cell adhesion molecules involved in synapse formation and the mechanisms regulating nerve terminal release of neurotransmitters. Through a combination of biophysical and biochemical techniques, work in Su¨dhof’s laboratory has led to the identification of several proteins that regulate SNARE-mediated neurotransmitter release, including synaptotagmin, complexin, and Munc18-1. In addition, his team has identified two trans-synaptic cell adhesion molecules, neurexins and neuroligins, which are crucial for proper synaptic function. He is currently focusing on understanding how these pre- and postsynaptic proteins control synapse formation and function during higher brain functions and how they can become impaired in neuropsychiatric diseases. Did you always want to be a researcher? Randy Schekman: My interest in science blossomed in junior high school with the annual science fair project. It deepened in college when I was introduced to experimental work in my freshman year at UCLA. At that point I lost interest in going to medical school and devoted myself to the lab, with decreasing attention to my coursework! I was greatly influence by Watson’s first edition of The Molecular Biology of the Gene and his autobiographical book, The Double Helix. The realization that one could use creativity, passion and hard work to probe the workings of the cell set me on the path to an academic research career. Thomas Su¨dhof: No – I initially wanted to be a practicing physician. It wasn’t until I performed research that I realized how much I loved it! How did you decide to focus on membrane assembly and vesicle trafficking? RS: I was influenced by my graduate advisor Arthur Kornberg, but even more by my best friend Bill Wickner who joined Kornberg’s lab fresh from graduate research in Eugene Kennedy’s lab at Harvard Medical School. My interests grew as a postdoctoral fellow in S.J. Singer’s lab at UCSD where I learned what was known about membrane traffic and where I first read George Palade’s brilliant work on the organization of the secretory pathway from his Nobel Lecture, which was published in Science the year he won the Nobel Prize. When I moved to UC Berkeley I resolved to work on this process in S. cerevisiae, motivated largely by the success of Lee Hartwell in using yeast Trends in Cell Biology, January 2014, Vol. 24, No. 1
Scientific Life: TrendsTalk genetics to dissect the cell division cycle. I was convinced that a combined genetic and biochemical approach to the secretory pathway in yeast would yield molecular mechanistic information that was not accessible using the then traditional tools of the morphological cell biologist. TS: All brain function depends on synaptic transmission. Neurotransmitter release initiates all synaptic transmission – it is half of the overall process, the other half being the sensing of the released neurotransmitters by postsynaptic receptors. I became interested in neurotransmitter release, which represents a vesicular trafficking event, because of its fundamental importance in brain function. I also became fascinated by it because of its unusual properties: it is extremely fast, occurring in a few hundred microseconds, tightly regulated by calcium, and highly plastic. What was your reaction when you received the call from Stockholm? RS: I was groggy from having just returned from Frankfurt the night before. Although I was aware that the Nobel decision would be announced that night, I had more or less given up on the idea that my work would be recognized by them. Therefore, the call really caught me by surprise, although by the time I stumbled over to the phone I was pretty sure who was calling. The feeling was surreal and it took a few moments to realize this was actually happening. TS: I was driving in the middle of Spain, somewhat lost, and my first thought was, ‘My God this is not true’ and then, ‘My God, this is wonderful.’ When you made your discovery, did you realize that it would have such a significant impact on the field? RS: When Peter Novick, one of my first graduate students, isolated our first secretion mutant – sec1 – I certainly had a strong feeling that this would allow us to develop molecular probes of the pathway and certainly lead to mechanistic insights. Nevertheless, I had no idea at the time that what we learned would have such direct relevance to the pathway in all eukaryotes, or that this knowledge could be applied in biotech companies to engineer the expression and secretion of important human proteins such as insulin. TS: My work consists not of a single discovery but of a series of observations that together explain different aspects of neurotransmitter release. There was no single paper or moment, but a series of revelations. Among them was synaptotagmin. The cloning already spawned the hypothesis that synaptotagmin is the calcium sensor for release (Perin et al., Nature 1990), and this was later supported by a series of biochemical experiments (Brose et al., Science 1992; Davletov and Sudhof, J. Biol. Chem. 1993) and studies on knockout mice (Geppert et al., 1994). The proof that synaptotagmin is the calcium sensor for release culminated with the demonstration that changing the calcium affinity of synaptotagmin changes the calcium affinity of release (Fernandez-Chacon et al., Nature 2001). A similar situation arose for the very important but overlooked question of how calcium channels localize next to release sites. When we cloned RIM we already knew that it would be important (Wang et al., Nature 1997), but it wasn’t until a series of knockout studies were performed that revealed how important RIM is for organizing neurotransmitter release (Schoch et al., Nature
Trends in Cell Biology January 2014, Vol. 24, No. 1
2002; Castillo et al., Nature 2002). This discovery culminated with the direct demonstration that RIM actually recruits calcium channels to the active zone, which was, in my view, an incredibly important observation (Kaeser et al., 2011). What do you think are the major outstanding questions in the field? RS: Many fascinating questions remain at the molecular mechanistic level about how vesicles form and how they are directed to their target and fuse with a target membrane. There is certainly enough to keep me interested and busy for the rest of my career. In my lab we are now working on the mechanism of autophagic membrane assembly and growth with a new cell-free system that recapitulates the first step in the biogenesis of the autophagosome. We are also keen to understand how large and unusually shaped cargo molecules such as collagen and lipoproteins are packaged into COPII transport vesicles that bud from the ER, how certain peroxisomal membrane proteins are packaged into transport vesicles that bud from the ER, how proteins destined for different cell surface domains are sorted at the trans-Golgi membrane, and how exosomal vesicles are generated at the cell surface or in multivesicular bodies. TS: Many questions remain open, some of which are detailed in a review I published in Neuron in November 2013. Fundamentally, we do not really understand the biophysics of fusion, we do not know which Sec1/Munc18like proteins are as essential for fusion as SNAREs are, and we still do not completely understand the mechanisms of presynaptic plasticity. There is so much to do! What is the best scientific advice that you have ever been given? RS: Focus. Perhaps this would have been my natural inclination, but what I learned from Kornberg was to focus on an important problem and remain dedicated to its solution. His specific advice was to pick an enzyme and study it. I didn’t follow that advice, but by his example I chose to study a pathway and, once the genetic approach showed promise, I reoriented my group to focus on that process using the genetic logic of Hartwell and the biochemical/enzymologic method I learned from Kornberg. TS: The best advice was not to worry about the journals I publish in, and not to worry about prizes. You have received the highest honor in the scientific community, what’s next? RS: Public education, and particularly public higher education, which was what made it possible for me to find my way in science. I am quite concerned that the next generation of aspiring young scholars from middle-class families may not have access to the great institutions that were available to me as I grew up. When I attended UCLA, beginning in 1966, I could pay the fees and room and board with income from a summer job. That is no longer possible, and families and students must go deep into debt to afford the same education that was given to me virtually free by the people of California. I intend to use whatever influence I have to make the case for continued investment in the University of California and in our other great public research universities. 7
Scientific Life: TrendsTalk TS: I really enjoy doing science and working with people, especially young people. Currently, most of my lab works on a completely unsolved problem related to autism and schizophrenia: how synapses are formed, specified, maintained, and restructured throughout life. I look forward to continuing this work. Finally, my hope is to contribute a little bit to the community, to the culture of science and university training, especially for the next generations.
8
Trends in Cell Biology January 2014, Vol. 24, No. 1
Figure acknowledgements: Randy Schekman and Thomas Su¨dhof, who with James Rothman shared the 2013 Nobel Prize for Physiology and Medicine. Photograph of R.S. (left) by H. Goren ß Howard Hughes Medical Institute; photograph of T.S. (right) ß S. Fisch. 0962-8924/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.11.006 Trends in Cell Biology, January 2014, Vol. 24, No. 1