Our understanding of the structure and function of muscle has known a number of outstanding electron microscopists who have contributed much to the current state of knowledge. On August 8, 2008, one of the most skilled of these died after a prolonged struggle against cancer. Mary Catherine Reedy’s contributions to the study of muscle structure and function are widely known in the muscle community where she was the collaborator of choice whenever thin section electron micrographs (EMs) of muscle tissue were needed.

figure a

Mary was an alumna of Rollins College in Winter Park, Florida, graduating with a Bachelor of Science degree in 1965. She later obtained a Masters degree in Biology from Georgetown University in 1967 where she was introduced to electron microscopy by Prof. George Chapman. After a short career in Marine Biology at the University of North Carolina in Chapel Hill, she joined the late Prof. James David Robertson’s laboratory at Duke University Medical Center in 1968. She remained at Duke University the rest of her scientific career and life. Mary mastered electron microscopy under the tutelage of one of the most demanding microscopists of his day, while applying her skills to a truly difficult problem, electron microscopy of membranes.

It was not until she married Michael Reedy (1973), gave birth to their son Carter (1974), and moved from Robertson’s lab to the Reedy lab in 1977 that her career in muscle microscopy began. The specimen of choice then as now was the asynchronous flight muscle of the large water bug Lethocerus sp. This insect flight muscle (IFM) has probably the best ordered sarcomeres in nature and was made famous by the ground-breaking Nature paper in 1965 by Mike Reedy, Ken Holmes and Richard Tregear describing the two extreme physiological states, relaxed and rigor. Up until the 1990s ushered in several studies of sudden length steps by millisecond-timed X-ray diffraction, along with the S1 crystal structure that suggested the tilting lever-arm refinement of the model, such IFM data formed much of the structural evidence behind the rowing crossbridge hypothesis, the major theoretical framework for understanding force production in muscle. IFM has since continued to produce the best 3D EM studies of different stages of muscle contraction.

Her ultramicrotomy and elegant microscopy of this already beautiful specimen showed Mary’s skills at their best. If IFM was the violin, she was the virtuosa. It was her micrographs, first appearing on the pages of the Journal of Muscle Research and Cell Motility in 1983, later in the pages and on the cover of Nature in 1984, and in the Journal of Molecular Biology in 1985, that cemented her reputation as probably the best thin section electron microscopist of her time, certainly in the muscle field. The 1983 JMRCM papers were notable for the many optically filtered micrographs used in correlating X-ray diffraction and electron microscope results. This EM and image analysis was largely done on a sabbatical leave with Mike in 1979–1980 at the EMBL in Heidelberg. Mike quickly admits that from the beginning Mary had a sharper eye for recognizing subtle differences in overall crossbridge lattice patterns: “I had been spoiled 15 years earlier by the easy distinction between rigor and relaxed lattices in IFM. Mary quickly leapfrogged my frustration and disappointment with the AMPPNP-induced lattice; she could see the differences despite the disorder, just by looking at the direct images on the EM phosphor screen, and her optical transforms and filtered images offered the proof of the pudding!” I am told that she could produce optical filtrations of electron micrographs faster than anyone could scan a micrograph and filter it digitally. This would not be possible today when so many images are collected digitally, but computer processing of micrographs was very different back then than it is now. The best electron micrographs of any non-rigor states of IFM so far produced were all obtained by Mary, beginning with a series of three papers in 1987–1988, on all three of which she was first author, and continuing through the collection of tomographic tilt series she selected and partnered in collecting with Jun Liu in my lab at FSU up through 2005.

My scientific collaboration with Mike and Mary began when I took up a faculty position in J. D. Robertson’s Anatomy Department in the summer of 1980. Mary, who made most of the specimens and collected all the micrographs for our collaborative work, would go to great lengths to get the images needed. For the first 3D reconstruction done on IFM in 1984, Mary manually tilted and filmed the dual axis tilt series, back long before it was either automated or even fashionable. For the 1985 JMB paper with Mike, she collected thin sections only 12 nm thick. Certainly sections 15 nm thick were routine for her. To even see anything in a 12 nm thick specimen required extraordinary staining protocols, developed by her and Mike. Particularly noteworthy from my perspective was her drive to find the images with very best orientation, needed in those days when spatial averaging was practically the only image processing we could do and electron tomography, the method of choice today, was in its infancy. Even her “rejects”, so named for their oblique orientations with respect to the fiber axis, were of such quality that we were able to convert them into 3D images using a novel reconstruction technique developed by Tony Crowther. However, the method would produce useful reconstructions only if the sections were very thin, something that few if any microtomists other than Mary were capable of collecting, let alone inclined to do.

When our collaboration moved to quick frozen, freeze substituted specimens, Mary’s skill became even more essential. With these specimens, obtained with Clara Franzini-Armstrong, Yale Goldman and Richard Tregear, good orientation combined with good cryo-preservation was hard to find and could only be located in a region about 3-μm deep into the freezing surface. It was only by sheer determination that she would seek, find and film tilt series images from the best oriented and preserved regions. From her efforts, we obtained the first 3D EM reconstructions of fast frozen contracting muscle that appeared in Cell in 1999. These images showed active crossbridges with a variety of lever arm orientations and a novel crossbridge motif. Much of her later work in this area is currently being written up; none of it would have been possible without someone of her skill and temperament to do the microscopy.

Besides Mary’s collaborative work on Lethocerus IFM, she contributed essential microscopic data on Drosophila flight muscle. Of her 71 publications identifiable via PubMed, 12 are on Drosophila IFM. She seems to have collaborated at one time or another with just about everyone working on Drosophila flight muscle. One of the more extraordinary papers was published in 1989 with the late Eric Fyrberg in Nature. An actin mutation retarded the incorporation of myosin and actin filaments into the sarcomere, resulting in disrupted Z-bands and misregistered filaments. Mary’s micrographs of the irregularly aligned sarcomeres showed myosin heads, in situ, binding normally to actin filaments in many areas. Occasionally, however, extended trains of rigor arrowheads of clearly opposite polarity formed along opposite sides of a single thick filament, showing that myosin heads could swivel 180° from their normal orientation to bind as “wrong-way-pointing” chevrons when forced to rigorize adjacent to “wrong-way-polar” actin filaments. Sellers and Kachar later showed that these interactions supported actin translation but at only 10% of normal speed. Another piece of work, of which she was particularly proud, was a complete morphological description at the electron microscopic level of time-staged pupal development of Drosophila flight muscle, done with Cliff Beall, that appeared as a pair of papers in Developmental Biology in 1993. Prior to that work, the process had to be inferred by comparison to myofibril formation in vertebrates and in IFM of larger, slower-to-hatch Calliphora.

Even outside of the field of IFM, Mary skillfully attacked difficult morphological problems in muscle research. A notable example where skill and determination obtained the necessary result was a study on the effects of autologous myoblast transplantation into scar tissue following a cardiac infarct, published in 1998 with Doris Taylor and William Kraus in Nature Medicine. Here Mary had to retro-adapt her thin-section methods to frozen serial sections from necropsied hearts, originally frozen for immuno- and histochemical mapping by light microscopy. These light-microscope maps located the small islands of striated skeletal muscle cells for electron microscopy within the mass of scar tissue. Electron micrographs showed that the skeletal myoblasts had not fused to form myotubes and instead had remained single myocytes with striated myofibrils and connected to each other via intercalated discs.

Prof. J. D. Robertson’s retirement as Chair of the Anatomy Department, and its reconstitution as the Department of Cell Biology by Prof. Michael Sheetz opened new opportunities for Mary to apply her technical skills to research problems completely outside of muscle research. It may surprise many in the muscle field that since the change in the department, 30% of her papers were on topics completely unrelated to muscle research. They covered quite diverse areas, from fast axonal transport in axon growth cones, published with Mike Sheetz in Nature to her most recent paper with Chris Nicchitta on mRNA localization, published in RNA. The axonal growth cone effort required new methods for precise micro-localization in thin-sections of laser-trapped vesicles and associated kinesin and dynein motors, whereby differential immuno EM identification of the motors was achieved using gold-tagged antibodies within the laser-trapped portion of the axon.

Much of her nonmuscle microscopy involved collaborations with colleagues Meg Titus (myosin I) and Arturo DeLozanne (myosin II), then at Duke, using Dictyostelium. High quality electron microscopy ascertained the detailed structure of the expressed protein within the cells. In one case, coexpression of myosin II LMM resulted in coassemblies of single-headed myosins which EMs were able to identify. Myosin I mutant phenotypes were notoriously difficult to demonstrate. Mary’s methodologies proved superb for Dicty ultrastructure and visualization of the cytoskeleton. EMs of controls were needed to demonstrate that the myosin was missing in the null mutants. This work helped build the case that single mutants of any null myosin I produced no detectable phenotype; only double mutants, knocking out two different myosin Is, would reveal a phenotype. Her Dicty work was not limited to nonmuscle myosins. Dietmar Manstein called upon Mary to analyze the ultrastructure of dynamin mutants to obtain detailed visual evidence of their effects on the cytoskeleton and membranes.

Because she was such a specialist and master of thin section electron microscopy, some might be tempted to describe Mary as mostly a technical virtuosa. This would do scant justice to her deep intellectual engagement in all her collaborative work. That she eschewed a Ph.D. degree doubtless hampered her academic development but not her scientific acumen. Her range of skill in electron microscopy was extensive, even to the point of completing and publishing a cryoEM helical reconstruction of actin filaments decorated with flight muscle S1 while on a sabbatical leave with Mike in Ron Milligan’s lab at The Scripps Research Institute in 2004. In all of the experiments on fast frozen contracting flight muscle to produce specimens for electron tomography and 3D imaging, she was involved at every stage of making specimens, including the mechanical experiments, not just the electron microscopy. Although sophisticated computing was not in her skill set, interpretation of the results certainly was. She spent many hours in my lab with a succession of postdocs going over IFM 3D reconstructions and quasiatomic models constructed from them. She was determined to know if the resulting fits were the best that could be obtained. She was involved in much of the recent synchrotron X-ray experimentation on flight muscle done with Mike and Tom Irving, the latest of which appeared this year in Proceedings of the National Academy of Science.

As anyone who has collaborated with her will attest, she formed her own independent opinion on any piece of work that involved her. If the field in which she was collaborating was new to her, she read the relevant literature and held her own in any argument. I can personally attest to the difficulty of moving her from an opinion that she held strongly. This was not mere stubbornness, just a desire to be sure that there was data to back up the assertions. She reveled in the give and take to determine what a series of experiments meant and how to present it. In all of our collaborative work, she was heavily involved in the writing and presentation. She reached a full level of professional acceptance when invited to write sole authored reviews in the flight muscle field. One observation, I think, says the most of her love of science. She continued her involvement in experiments until the last few months of her life and when she could no longer go into the lab, worked on manuscripts right up to the final few weeks of her life when pain control medicines limited her attention span. In her last 10 days, she insisted that Mike join his team at Argonne-APS taking synchrotron X-ray diffraction “movies” of glycerinated IFM performing sinusoidally driven oscillatory “work-loops”. When he returned 6 days later and told her of the team’s highly successful beamtime, her wide and wonderful sudden grin said it all: “How wonderful! I only wish I’d been able to go along!”

Mary had a well-rounded life. She minored in art as an undergraduate, painted watercolors and collected art objects throughout her life. Research gave her many opportunities for extended stays abroad. She made many prolonged visits to Italy, from her days studying octopus brain with J. D. Robertson at the Stazione Zoologica in Naples to more recent visits with Mike to Vincenzo Lombardi’s muscle physiology lab in Florence. She loved Italy and everything Italian and leaves her home well-decorated with many Italian artifacts. She always spoke fondly of her working visits to EMBL that began with a sabbatical in 1978–1979 followed by numerous working visits through 1994 with Belinda Bullard to perform immunoEM on flight muscle. I introduced her to La Jolla, CA in 2003, a place she fell in love with immediately, so much so that she and Mike spent a semi-sabbatical there in 2004 and loved every minute of it. Gardening was also one of her passions; in her backyard garden, you could feel completely isolated from the world’s problems by tall ligustrum hedges and still taller bamboo screens. There you could forget all about living in a small city. She loved the Biophysical Society and the whole muscle field, and was loved in return, thanks to her brightly positive personality and broad interest in the work of so many others. Meetings will not be nearly as interesting from now on without her spark. Mary trained few protégé’s so it may be some time before electron microscopy sees a person with such highly refined skills combined with a determined temperament to get the result. She illuminated all of our lives with her passion and will be sorely missed by friends and colleagues around the world.