Supplementary Material From Singer Lab Publications:

The scientific community lost one of its most gifted and productive members on March 18, 1997 when Fred Fay suffered a fatal heart attack. Fred was born in New York City, and was educated in its public school system. Upon graduation from the Bronx High School of Science in 1961, he enrolled at Cornell University where he majored in Chemistry and was elected to Phi Beta Kappa. His love and talent for science were evident early, not only in the schoolboy experiments conducted in the family apartment, but also in the summers of his undergraduate years when he worked as a lab assistant at the Sloane-Kettering Institute. Fred began his formal scientific career in his junior year at Cornell when he joined the lab of Dr. R. Blake Reeves. With Reeves Fred first studied the effects of low barometric pressure on the synthesis of a myoglobin-like oxygen binding pigment in a marine arthropod. This was followed by a study of B12 uptake in a protozoan which led to Fred's first scientific publication: (Reeves RB and Fay FS. Cyanocobalamin (Vitamin B12) uptake by Ochromonas malhamensis. Am. J. Physiol. 210:1273-1278, 1966.). Reeves had recently arrived at Cornell after completing his doctoral and post-doctoral training in the Physiology Department at Harvard Medical School. As is so often the case, Reeves referred his star pupil to his mentor, Dr. John R. Pappenheimer, and upon graduation from Cornell, Fred enrolled in the Medical Sciences Program at Harvard to begin his doctoral studies in physiology.

Pappenheimer had longstanding interests in regulation of respiration and in oxygen sensing mechanisms, and at the time was studying the contribution of the ionic composition of the cerebrospinal fluid to the regulation of respiration in goats. It was quite natural in this environment for Fred's interest to be tweaked by the question of how tissues sense and respond to changes in oxygen tension. He examined the anomalously high rate of oxygen consumption in the carotid body for clues to its oxygen-sensing function. These studies provided the first accurate data on oxygen consumption by this tissue which in fact consumed oxygen at less than twenty per cent of the previously accepted rate. To complete his thesis project he went on to study contractile responses of the guinea pig ductus arteriosis to increased oxygen availability. This latter research kindled the interest in the physiology of smooth muscle that was to play a central role in his research career. These early studies also foreshadowed other themes that became prominent in all of his subsequent work. Fred had a need to see what was happening in his experiments. His thesis and the resulting papers beautifully portrayed the morphology of his preparations at the macroscopic and microscopic levels. Studies of both the carotid body and the ductus arteriosus required the custom design and fabrication of suitable apparatus and carried the imprimatur of the inventiveness and skill that characterized all of his later work.

After receiving his Ph.D. in 1969, Fred stayed on at Harvard to continue work on the guinea pig ductus arteriosus. These studies showed all of the elegance and clarity of thought that were to become the hallmark of Fred's science. He found that tension in the ductal wall increased in response to increased partial pressure of oxygen just as well when oxygen was present only in the perfusate within the vessel or when present only in the perifusate bathing the outer adventitial surface that contained nerve endings and mast cells. Because equal, but oppositely directed gradients of PO2 produced equivalent contractile responses, he concluded that the smooth muscle itself acted as the oxygen sensor and contracted in a stepwise fashion (Fay FS Guinea pig ductus arteriosus I. The cellular and metabolic basis for oxygen sensitivity, Am J Physiol 221:470-479, 1971). He later showed that the trigger for this response is a unique cytochrome and demonstrated that the ductus could be made to contract by light of the appropriate wavelength if the cytochrome had first been inactivated by carbon monoxide in the dark. Virtually all of the themes that were to characterize his later work were already germinating in these studies: the morphologic studies, particularly associated with the irreversibility of ductal closure and the questions raised with regard to coupling between metabolism and electrical activity and contraction, and the stepwise contractile behavior of "a population of smooth muscle cells of the single-unit type".

Meanwhile, forty miles to the west final plans were taking shape to open the UMass Medical School. Fred was recruited as an Assistant Professor of Physiology, and was in Worcester to greet the first class of medical students when they arrived in September of 1970. He was intimately involved in all of the planning, organizing, and just plain hard work that goes into starting a new department. Much of the credit for whatever success the Department now enjoys must go to him. With characteristic energy and drive Fred immediately set up shop in a small lab in the converted tobacco warehouse that was to be the temporary home of the medical school for the next three and a half years. Here he continued his studies on the ductus arteriosus and on the morphologic basis for contractility of smooth muscle in collaboration with Peter Cooke. He also began to lay the groundwork for indepth studies of smooth muscle physiology.

At that time understanding of how smooth muscle contracts was meager at best. All that was known was derived from studies of multicellular preparations with all of the complexities attributable to heterogeneity of cells and extracellular matrices. Fred began to rectify that situation with characteristic insight and boundless energy. He set about trying to develop a homogeneous population of isolated muscle cells to define the contractile properties of smooth muscle cells themselves. His first attempts were with the intestinal muscles of a large salamander, Amphiuma sp., which had unusually large smooth muscle cells. Unfortunately, obtaining a dispersed cell preparation that maintained normal contractility was no simple matter. As he struggled with Amphiuma, Bagby and colleagues (Bagby, R., Young, A., Dotson, R., Fisher, B., and McKinnon, K. Contraction of single smooth muscle cells from Bufo marinus stomach. Nature 234:351-352, 1971.) published their successful preparation of isolated smooth muscle cells from the stomach of Bufo marinus. Fred was quick to exploit this preparation and undertook what are now his classic studies of smooth muscle physiology.

If there is any one aspect of Fred's career that stands out, it was his use of the single smooth muscle cell preparation. With it, Fred explored virtually every aspect of smooth muscle physiology and molecular anatomy at the cellular and subcellular level. Fundamental contributions were made concerning the organization of the contractile apparatus and how it is turned on and off in response to various physiological signals. This unique preparation was not only a model system for smooth muscle, but also a proving ground for Fred's views of cell biology. Working with a medical student, Claudio Delise, Fred showed with scanning electron microscopic images that when a smooth muscle contracts large +blebs+ appear on the plasma membrane and proposed that the contractile machinery attaches at specialized regions of the plasma membrane. The now classic article describing these findings (Fay, FS and CM Delise. Contraction of isolated smooth muscle cells-structural changes. Proc Natl Acad Sci USA 70:641-645, 1973) was his third most cited paper.

With another medical student, Peter Canaday, he devised an ultrasensitive force transducer. He then developed the techniques for attaching a single smooth muscle cell to the transducer and recorded tensions of a few micrograms when the cell contracted (Fay, FS. Isometric contractile properties of single isolated smooth muscle cells. Nature 265:553-556, 1977). With this new technology, Fred showed that, in contrast to striated muscle or the multifiber smooth muscle preparations used by all previous investigators, the tension of a relaxed smooth muscle cell is virtually independent of its length. These studies highlighted the major contribution of the connective tissue elements to the mechanical properties of smooth muscle tissues and thus confirmed the critical importance of studying isolated smooth muscle cells for understanding of smooth muscle physiology. In these studies, Fred also made the unique observation that there was a delay between the excitatory stimulus and the onset of force generation. He suggested that the delay was due either: 1) to a delay in coupling of the changes in membrane potential to the change in intracellular Ca2+ ; or, 2) that Ca2+ is not rate limiting and that Ca2+-dependent smooth muscle myosin regulatory processes are slow.

The importance of being able to measure intracellular calcium concentrations ([Ca2+]) was evident, and when he became eligible for his first sabbatical leave, Fred decided to work with Dr. Stuart Taylor at the Mayo Medical School to learn how to measure intracellular Ca+2 using aequorin as a calcium indicator (Fay, FS, HH Shlevin, WC Granger and SR Taylor. Aequorin luminescence during activation of single isolated smooth muscle cells. Nature280:506-508, 1979). He microinjected aequorin into individual smooth muscle cells and discovered that upon stimulation [Ca2+] rises rapidly and remains above basal levels. Therefore, Ca+2 delivery to the regulatory proteins is not rate limiting. Rather, it is the myosin light chain kinase regulatory pathway that is relatively slow, thus generating the observed delay between stimulus and contraction. This finding inevitably generated the next question: what Ca+2 handling processes might govern the decay of [Ca+2]? Its answer would have to wait for the development of new techniques, and he would revisit this concept 20 years later.

Fred loved to hypothesize about molecular mechanisms. He loved to look at a data set and turn it around and around in his mind to uncover every facet. He would challenge his colleagues or fellows to come up with every possibility that might explain the data and then rigorously weigh every potential explanation until only the one or two most plausible survived. He wasn't shy in going out on a limb to propose unique mechanisms. A good example of this is seen in his second most cited paper (Scheid, CR, TW Honeyman and FS Fay. Mechanism of b-adrenergic relaxation of smooth muscle. Nature 277:32-36, 1979). Fred and his coworkers proposed that b-adrenergic relaxation of smooth muscle cells was a result of cAMP-dependent stimulation of the Na+/K+ ATPase pump which, in lowering intracellular sodium would, in turn, stimulate the Na+/Ca2+ exchanger, with the final outcome being a reduction in [Ca2+]i. It wasn+t until fourteen years later, and after his lab had developed the digital imaging microscope, that Fred was able to expand on this idea. Fred reasoned that if b-adrenergic relaxation involved both Na+/K+ ATPase activity coupled to the Na+/Ca+2 exchanger, these two entities might be physically co-localized within the smooth muscle cell. By pushing fluorescence detection coupled to the digital imaging microscope to its technological maximum, Fred and his colleagues also pushed back the limits of spatial resolution. Using immunohistochemical methods, they demonstrated that the Na+/K+ ATPase and the Na+/Ca+2 exchanger were indeed co-localized in the plasma membrane and were restricted to regions that were distinct from those membrane regions that were specialized for mechanical force transmission (Moore EDW, EF Etter, KD Phillipson, WA Carrington, KE Fogarty, LM Lifshitz and FS Fay. Coupling of the Na+/Ca2 + exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle. Nature365:657-660, 1993). With McCarron and Walsh, Fred then went on to show that Na+/Ca2+ exchange regulates cytoplasmic calcium in smooth muscle.

Efforts to understand how smooth muscle contracts had been hampered by the lack of any technology that would allow Fred to look inside the cells and see what was happening molecule by molecule. He therefore set out to devise optical instruments to do just that. The development of the Digital Imaging Microscope (DIM) was critical to the next steps in Fred's scientific career. He assembled an extraordinary team of physicists, mathematicians, and computer scientists who together created powerful and innovative optical technology that placed the Imaging Group at UMass at the forefront of biomedical imaging at the cellular and subcellular levels. Over the past ten years the digital imaging microscope has evolved to keep pace with the complexity of the scientific problems to which it has been applied. For example, the studies of the distribution of the Na+/K+ ATPase and the Na+/Ca2+ exchanger required three dimensional images of immunolabeled fixed cells. A single data set of multiple focal planes within a cell was taken over a period of several minutes. Such a leisurely time course clearly was inadequate to tackle the rapid dynamic changes that occur in living cells. In the early 1990s a high speed instrument was developed that used laser illumination, high speed piezo electric focus, and a conventional scientific CCD camera partially masked so that different portions of its imaging area could be used to take images a few milliseconds apart. This instrument was used to measure changes in the membrane potential of individual mitochondria which were in motion at speeds of up to one micron per second in neuroblastoma cells (Loew, L.M., Tuft, R.A., Carrington, W.A., and Fay, F.S. Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria. Biophys. J. 65:2396-2407, 1993) and was fast enough to permit study of Ca2+ gradients in the first fifteen milliseconds of systole in cardiac myocytes (Isenberg, G., Etter, E.F., Wendt-Galliteri, M., Schiefer, A., Carrington, W.A., Tuft, R.A. , and Fay, F.S. Intrasarcomere [Ca2+] gradients in ventricular myocytes revealed by high speed digital imaging microscopy. Proc. Natl. Acad Aci USA 93:5413 5418,1996). Even greater speed was achieved recently with a high speed CCD camera developed for the Strategic Defense Department, and made possible Fred's last major work in which he studied the relationship between calcium "sparks", which are localized quantal releases of Ca2+, and spontaneous transient outward potassium currents (Kirber, M.T., Etter, E.F., Singer, J.J., Walsh, J.V. Jr., and F.S. Fay. Simultaneous 3d imaging of Ca2+ sparks and ionic currents in single smooth muscle cells. Biophys. J. 72:(2)part 2 A295, 1997).

Fred's involvement in his science was far reaching. Although a skilled machinist was available, if a custom part for the microscope had to be made quickly, Fred would use the milling machine himself to build the part. He, as the biologist, did not simply delegate the application of complex physical and mathematical concepts to his team of experts, but remained an interactive contributor to the incorporation of these ideas into workable instruments. Inherent in the workings of the digital imaging microscope was the ability to account for fluorescence from out of focus optical planes. This was no small feat. Fred thoroughly understood the physics and mathematics behind these sophisticated de-blurring techniques. His most trusted reference in this regard was the "Feynmann Lectures".

Fred was among the earliest investigators to avail themselves of Fura-2 as a Ca2+ indicator. He quickly marshalled the resources to obtain the necessary equipment to make ratiometric fluorescent measurements in his isolated smooth muscle cells. Before long he had the requisite technology incorporated into the digital imaging microscope. These technical advances led to Fred's most cited paper, now an official citation classic (Williams DA, KE Fogarty, RY Tsien and FS Fay. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using fura-2. Nature318:558-561, 1985). But Fred wasn't satisfied with global measurements of Ca+2. He developed the ability to monitor intracellular calcium with both high spatial and temporal resolution. With this technique, Fred and his fellows demonstrated that Ca+2 gradients existed within the cell with higher [Ca+2]i within the nucleus and at the sarcolemma, presumably due to Ca+2 stores within the sarcoplasmic reticulum. Fred demonstrated that cytoplasmic Ca+2 was about 140 nM under resting conditions and about 700-800 nM under activated conditions. These data indicated the exquisite sensitivity of the contractile apparatus for Ca+2. The specialization of the membrane for calcium handling suggested that there may be significant differences between measurements of global cellular calcium compared to those close to sites of calcium release and storage. This is most evident in Fred's most recent work where he utilized membrane-bound calcium indicators (Etter EF, A Minta, M Poenie and FS Fay. Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18. Proc. Natl. Acad. Sci USA 93:5368-5373, 1996) which were capable of reporting local calcium changes adjacent to the plasma membrane.

Fred contributed not only to our understanding of the contractile machinery, but also of the regulatory processes that govern smooth muscle contraction. A major question in this field was whether or not activation in smooth muscle contained a calcium-dependent actin filament linked regulatory system, analogous to that in striated muscle. In a novel experiment, Fred and coworkers microinjected the catalytic subunit of myosin light chain kinase into single smooth muscle cells and observed that, in the absence of changes in intracellular calcium, phosphorylation of the regulatory light chain on smooth muscle myosin was sufficient to elicit a contraction. These data refuted the necessity for an actin-linked regulatory system but did not rule out a possible modulatory role for such a system (Itoh T, M Ikebe, GJ Kargacin, DJ Hartshorne, BE Kemp, and FS Fay. Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells. Nature 338:164-167, 1989).

Fred was a renaissance scientist whose interests were not limited to smooth muscle. He had an ongoing interest in chemotaxis and the relationship between changes in intracellular calcium and cellular motility (RA Brundage, KE Fogarty, RA Tuft and FS Fay. Calcium gradients underlying polarization and chemotaxis of eosinophils. Science 254:703-706, 1991). His lab also examined a variety of topics that explored the relation of molecular distribution to cell function. Studies either initiated in his lab or conducted collaboratively with other scientists at UMass or around the world included explorations of the association of hexokinase with mitochondria, the distribution of precursor messenger RNA in the nucleus, the codistribution of polyA RNA with microfilaments, the localization of a yeast splicing factor in subnuclear domains, the migrations of MAP kinase, and intracellular trafficking of growth factor receptors.

Fred was a superb scholar and teacher. He was not satisfied with proving to himself that he understood the physical principles behind the optical techniques that he incorporated into development of optical instruments. Fred believed that the mark of knowing whether he really understood a concept was to then try and teach it to someone else. He therefore designed a successful graduate course in "Optical Methods" which served as a great forum for presenting established ideas and developing new ones. Fred also was an active participant in the Medical Physiology Course given each year to the first year medical students. Over the years he taught a wide range of physiological topics, inevitably coming up with new insights and approaches to solidly established findings. Fred always had the patience to help his fellows learn the art of public presentations. He would videotape his fellow's job interview seminars and spend hours going over them point by point until he was satisfied. His standards were high and he brought out the best in all who worked with him.

Fred will be remembered by his students and colleagues in Worcester and on every continent for more than his science, for he was a very special human being. His home, his heart, and his mind were open for all to share, and his wit and energy brought joy to all who were privileged to know him. He worked hard and played hard. He touched many lives, and left them all better for the contact. He took great pleasure and pride in his wife Madeleine, his children, Andrew, Nicholas, and Isabel and his grandchildren, Sarah, David, and Julia. He also derived great joy from the scientific offspring who worked in his lab. The list includes seven medical students, five graduate students, twenty-three postdoctoral fellows, and six senior scholars who spent their sabbatical leaves with him. Many of his former students and fellows have gone on to build impressive careers for themselves, and their science will always bear Fred's indelible imprint. Fred died far too soon, but we can take some comfort in the knowledge that he packed a great deal into his all too short lifetime. In all he published over 125 papers and reviews in the most highly critical and respected journals. His creativity resulted in the award of eight patents, and his technological innovations in optical methods paved the way for him and future generations to "boldly go where no one has gone before".

H. Maurice Goodman, Ph.D
Department of Physiology
University of Massachusetts Medical School
Worcester, Massachusetts

David Warshaw, Ph.D.
Department of Molecular Physiology and Biophysics
University of Vermont Medical School
Burlington, Vermont