Background and Scientific Scope of NASA Programs
The National Aeronautics and Space Administration (NASA) manages research programs in two areas of the rapidly expanding field of biotechnology: protein crystal growth and cell science. The protein crystal growth work focuses on using microgravity to produce higher quality macromolecular crystals for structure determination and on improving understanding of the crystal growth process. The cell science work focuses on basic research that contributes to understanding how the microgravity environment affects the fundamental behavior of cells, particularly in relation to tissue formation and the effects of space exploration on living organisms. The National Research Council 's Task Group for the Evaluation of NASA's Biotechnology Facility for the International Space Station was formed to examine and evaluate the use of the International Space Station (ISS) as a platform for research in these two areas.
The logistics associated with space-based research pose a unique challenge to investigators. Strict limitations on the weight and volume of equipment, long planning times, and the remoteness of experiments from the scientists who design them all make research in microgravity —on a space shuttle or on a permanent space platform—very difficult. The reward is an opportunity to work in an environment unavailable on the ground and to examine issues related to gravitational forces, a fundamental part of life on Earth.
The primary motivation for the construction of the ISS has been not the tackling of specific scientific questions but rather the political and cultural need of this country, and perhaps the world, to explore the universe and push at the boundaries of what is humanly achievable. This motivation is not a technical one, and the task group was not set up to evaluate or comment on whether the ISS should be built. Instead, it assumed that assembly of the ISS, having started, would be completed, and focused on how this new research platform could be most effectively utilized. This report therefore discusses whether science in a microgravity environment can advance the fields of macromolecular crystallography and cell science, and if it can, in what areas the advances will most likely occur. The task group discusses issues related to the instrumentation needed to conduct effective research on the ISS and, finally, comments on human factors—how NASA can communicate results and opportunities effectively and enable investigators to design and execute the best possible experiments for the ISS. It offers a variety of recommendations and suggestions for improving the NASA biotechnology research program, and it believes that these changes are necessary if the NASA program is to fulfill the potential for scientific discovery and impact outlined in this report.
PROTEIN CRYSTAL GROWTH
During the 1990s, there was explosive growth in the number and complexity of macromolecular structures being determined by X-ray crystallography, as evidenced by the exponential increase in the number of structures published and submitted to the Protein Data Bank. This growth has been made possible by the convergence of a large number of new technologies, including the following:
Improved systems for cloning and expressing wild-type and mutant proteins;
Improved protein and nucleic acid purification techniques;
Immortalization of crystals by cryogenic freezing;
Very high brilliance X-ray synchrotron sources;
Fast, accurate area detectors with high dynamic range;
Superfast, inexpensive computers; and
Readily available software packages for data acquisition and reduction, phasing, and refinement.
For the most part, however, protein crystallization is done in much the same trial-and-error manner it was a decade ago, albeit with somewhat less tedium since the introduction of reagent kits and the growing use of automated systems. It is still more art than science. NASA, to its credit, has sponsored a large number of ground-based research projects aimed at understanding the fundamentals of the crystallization process. These included investigations of depletion zones around growing crystals (McPherson et al., 1999), studies of defect formation during protein crystal growth and the effects of these defects on diffraction resolution (Dobrianov et al., 1998, 1999), and analyses of predictors for protein crystallization using light-scattering measurements (Kao et al., 1998; Ansari et al., 1997). 1
These studies of the crystallization process have occasionally included flight components (McPherson et al., 1999). However, one of the main goals of NASA's program on crystallization in the microgravity environment has been the growth of crystals in space that are of better quality than those available on the ground. In this report, the task group focuses on evaluating the results of the program's effort in this area to date, commenting on the hardware available and in development for future work on the ISS and offering suggestions for improving the project selection process and NASA's outreach to the scientific community.
The Significance of Crystallographic Resolution Limits
The determination of macromolecular structures by X-ray crystallography at a level of detail sufficient for the construction of reliable atomic models requires crystals that diffract X rays to Bragg spacings of 3.5 Å or better. The minimal Bragg spacing to which diffraction measurements can be obtained, loosely referred to as the resolution of the crystallographic analysis, limits the accuracy of the resultant structure in two ways. First, the resolution of the analysis places a limit on the structural features that can be directly visualized in electron density maps calculated using the X-ray data. A resolution of at least 3.5 Å is required to see structural elements in proteins, such as alpha-helices or beta-sheets. Second, once an initial atomic model has been constructed, the resolution of the analysis determines the accuracy with which the parameters of the atomic model can be refined. Positional coordinates in a refined macromolecular structure are determined much more precisely than the resolution of the analysis would indicate. When stereochemical constraints are used in the refinement, such as information about the bond lengths between atoms, the precision of a protein structure typically is approximately 0.5 Å for an analysis carried out at 3 Å resolution and is better than 0.1 Å for a 1.5 Å resolution analysis. In the relatively rare cases where data to better than 1 Å are obtained, individual hydrogen atoms can often be distinguished and the disorder within the protein structure can be described in detail.
These are a few examples of the projects under way; a complete list and description of NASA-funded projects in protein crystal growth can be obtained on the Web at <http://peer1.idi.usra.edu/peer_review/taskbook/micro/mg99/mtb.cfm >.
Although a protein crystallographer will always strive to carry out the crystallographic analysis at the highest achievable resolution, the minimal acceptable resolution for a particular crystallographic analysis depends on what questions are being asked. Where the general fold of the protein chain is desired, an analysis at 3.5 Å may suffice to determine the protein structure. However, at this resolution the orientation of hydrogen bonding groups is not well determined, and detailed questions regarding the structural architecture of the protein cannot be answered reliably until a resolution of ~2.5 Å or better is achieved. The precise calculation of the energetics of ligand binding or intermolecular interfaces requires structure determination carried out to an even higher resolution, making possible the mapping of ordered water molecules and an accurate description of hydrogen bonding geometries, and this mandates a resolution of 2.0 Å or better. The most accurate protein structure determinations are carried out at a resolution of 1.5 Å or better.
The intrinsic resolution of a protein crystal can be thought of as arising from two factors. One is the mosaicity, a parameter that is a measure of the misalignment between small coherent blocks of individual molecules within the larger crystal. While crystals that are highly mosaic may diffract to high resolution, the high mosaicity leads to a broadening of the diffraction spots, which can complicate or even foil their measurement. The other crystal characteristic that affects resolution is the Debye-Waller factor, also known as the overall temperature factor, which reflects disorder and mobility within the individual molecules that make up the crystal. Many protein molecules that are of interest today, particularly those that are studied in the form of relatively unstable complexes, are expected to have intrinsically high Debye-Waller factors, limiting the resolution of the resulting diffraction pattern. In such cases, if the size of the perfectly aligned mosaic blocks can be increased, the resulting increase in the sharpness of the diffraction pattern can effectively improve the resolution of the diffraction pattern. In such situations, if growth in the microgravity environment produces crystals with larger mosaic blocks (lower mosaicity), then there may be a significant improvement in the quality of the diffraction measurements. These added levels of detail would enable researchers to see the functional groups and water molecules and thereby more fully understand the interactive mechanisms of macromolecular assemblies.
Today's high-energy synchrotron sources have, in general, eliminated crystal size as the key factor in increasing the diffraction resolution limit. This was not the case when the space crystallization program began, in the mid-1980s. Although the misconception that size is crucial may persist at NASA, scientists today are interested in crystallization methods that provide higher quality crystals, where quality is measured by disorder and mosaicity. Therefore, a well-ordered crystal of average dimensions (around 30 to 50 µm) is all that is needed for effective diffraction studies. Synchrotron technology continues to improve, and the target crystal size may decrease even further before the ISS is completed. Crystal quality, rather than crystal growth, is thus the primary focus of the biological macromolecular research community. The only exceptions are during initial efforts to nucleate protein crystals and when preparing samples for study via neutron scattering.
Goals and History of the NASA Protein Crystal Growth Effort
The current goals of the protein crystal growth efforts funded by NASA's Microgravity Research Division are as follows:
Understanding the fundamental factors influencing macromolecular nucleation and growth;
Elucidating which factors may benefit crystal growth in the microgravity environment;
Growing significantly improved crystals in microgravity for structure determinations;
Determining the potential of microgravity to solve more complex and challenging crystallization problems, such as integral membrane proteins, glycoproteins, and lipoproteins; and
Developing technologies and methodologies such as automation and monitoring equipment that would improve the crystallization process on Earth as well as in space.
The program began with exploratory efforts to grow macromolecular crystals in space in 1985. From that time through October 1999, experiments relating to the crystallization of biological samples were carried out on 43 NASA missions. This has resulted in a total of 185 different proteins and other biological macromolecular
assemblies being studied. Overall, the results from the program so far are inconclusive. In many of the cases that have been listed as successful, the improvements obtained were incremental advances in resolution rather than substantial increases. In certain cases, such as the result reported for T³R3f insulin, where a sizeable improvement in resolution appears to have been obtained by the application of microgravity (Smith et al., 1996), the impact of the work is lessened by the fact that a considerable amount of structural information was already available for insulin. Consequently, the enhanced resolution observed for the space-grown crystals did not enable a distinctly new body of information to be obtained. Due to both the limited number of experiments and the type of proteins for which significant improvements were noted, the impact of microgravity crystallization on structural biology as a whole has been extremely limited. At this time, one cannot point to a single case where space-based crystallization efforts produced a crucial discovery leading to a landmark scientific result. In addition, the difficulty of mounting simultaneous efforts to produce the best possible crystals both on the ground and in space has limited the ability of researchers to make the comparisons between microgravity and Earth crystals that would be necessary to demonstrate that the microgravity environment can produce superior crystals.
Finding: The results from the collection of experiments performed on microgravity's effect on protein crystalgrowth are inconclusive. The improvements in crystal quality that have been observed are often only incremental, and the difficulty of producing the appropriate controls limit investigators ' ability to definitively assess if improvements can be reliably credited to the microgravity environment. To date, the impact of microgravity crystallization on structural biology as a whole has been extremely limited.
Despite the lack of impact of microgravity research on structural biology up to now, there is reason to believe that the potential exists for crystallization in the microgravity environment to contribute to future advances in structure determination. All research on protein crystallization in space so far has been done under suboptimal conditions. Most of the work has been done on fairly short space shuttle flights, with a few experiments occurring on the Russian Mir space station. The crystallization work on the space shuttle has been restricted to a matter of days, which is not enough time in most cases to complete the crystallization process, especially in space, where crystals appear to nucleate and grow more slowly. Except for space shuttle missions devoted exclusively to microgravity research, the environment on the space shuttle has not been noise- and vibration-free. No mechanism has been provided to stabilize the crystals that do grow and to protect them from the stresses of reentry. In general, the ability to visualize crystal growth in space has been extremely limited, preventing investigators from determining if flawed crystals examined after landing had failed to grow well in space or if crystals with good morphology had indeed been grown but later had been damaged during reentry. The irregular schedules of shuttle missions and the long lead times have made it difficult for scientists engaged in extremely competitive structural analyses to seriously consider participation in the shuffle-based crystallization experiments. Long delays between shuttle flights has meant that lessons learned from one flight are often not translated into improved experiments on a subsequent flight. The slow and uncertain progression of experiments on the space shuttle has disconnected them from the even more rapid tempo of contemporary protein crystallography research.
Results to Date: Examples of Successful Experiments and the Importance of Defining Controls
Despite these limitations, it is conservatively estimated that for 36 of the 185 different proteins and other biological macromolecular assemblies that have been studied in space, the resolution of the crystallographic analysis was better than that of the best ground-based results available at the time. The proteins whose resolution improved in space range from well-understood test cases, such as lysozyme, to proteins that present significant challenges for contemporary structural biology, such as the EcoRI-DNA complex, the nucleosome core particle, and the epidermal growth factor receptor. Enhanced resolution has also been obtained for proteins of importance for drug design, including the HIV protease complexed with lead compounds and the influenza neuraminidase.
While this list of improved crystals is certainly tantalizing, it is difficult to draw concrete conclusions from the limited data available, for the reasons discussed in the previous section. Below, the task group describes in detail
four of the positive results. These cases were selected because the researchers were able to cleanly compare crystals grown in space to the best crystals produced using ground-based systems and because the improvements in resolution were substantial. The four examples are lysozyme, canavalin, satellite tobacco mosaic virus (STMV), and insulin. While the proteins in these experiments are not in themselves necessarily of great biological significance, the studies are important because of what they indicate about the potential benefits of crystal growth in space.
Lysozyme, a workhorse in the field of protein crystal growth, yields crystals in space that have much better properties than those of crystals grown on Earth. Analysis of tetragonal lysozyme crystals grown on two space shuttle missions showed, impressively, that the mosaicity of the crystals was improved by factors of 3 or 4 over that observed for lysozyme crystals grown on Earth (Snell et al., 1995). The observed reduction in mosaicity is a very significant improvement, because it can allow the measurement of very weak reflections that would otherwise be too broad to be observed over background. Although crystals of lysozyme with very low mosaicity can occasionally be obtained on Earth, only about 1 in 40 of them have properties comparable to those of the crystals grown in space.
STMV is a small icosahedral plant virus, consisting of a protein shell made up of 60 identical protein subunits of molecular weight 14,000. The crystallization of STMV has been studied extensively on Earth. Its crystallization in microgravity was investigated during two space shuttle missions, in 1992 and 1994. Using a liquid-liquid diffusion technique with careful temperature control (in an experimental setup known as CRYOSTAT), remarkably large crystals of STMV were obtained (Day and McPherson, 1992). In most cases the crystallization chambers contained large single orthorhombic crystals, the largest of which measured 1.5 mm in length and 1.0 mm in both of the other directions. These crystals are about 10 times larger in volume than the largest crystals of STMV previously grown in ground-based laboratories. Particularly noteworthy is the fact that, in contrast to the crystals grown on Earth, the crystals grown under microgravity conditions were visually perfect, with no striations or clumping of crystals. Furthermore, the X-ray diffraction data obtained from the space-grown crystals was of a much higher quality than the best data available at that time from ground-based crystals. The average intensity of the diffraction measurements relative to the standard deviation was seen to be increased substantially over the entire resolution range, resulting in nearly 50 percent more X-ray data than had previously been available. STMV also crystallizes on Earth in a cubic crystal form that diffracts poorly; at the time of the 1994 shuttle flight, the best available ground results gave only about 6 Å resolution (Koszelak et al., 1995). Cubic crystals of STMV obtained on board the space shuttle were 30 times larger than those obtained on Earth. These crystals diffracted X rays to 4 Å resolution, a significant improvement over the ground-based crystals.
Canavalin is a plant storage protein from the Jack Bean and is a trimer of three identical subunits of molecular weight 47,000. It can be crystallized reliably on Earth, which has made it one of the proteins commonly used for crystallization studies. As with STMV, large crystals of canavalin were obtained in space (Koszelak et al., 1995). Visually perfect rhombohedral and hexagonal crystals of canavalin with edges >1 mm in length were obtained in large numbers, with significantly better diffraction properties than those of crystals grown on Earth. For the rhombohedral crystals, the diffraction limit was extended from 2.6 Å to better than 2.3 Å. The improvement in resolution for the hexagonal crystals was more impressive: it went from about 2.7 Å (Earth) to nearly 2.2 Å (space). In both cases the total number of useful X-ray measurements essentially doubled.
The crucial human hormone known as insulin consists of two chains, an A-chain consisting of 21 residues and a B-chain consisting of 30 residues. Insulin aggregates to form hexamers, which undergo allosteric transitions between R and T states. The switching between the R and T states is altered by the presence of particular ions and organic molecules, and there is interest in identifying additives that would stabilize the R-state over the T-state, which would lead to insulin preparations with greater stability. To this end, high-resolution crystallographic analyses of insulin are being carried out. This project has led to a particularly clean comparison between the results of ground-based and space-based crystallizations of a protein. Human T³R3f insulin obtained from a single source was crystallized on Earth and also in microgravity using batch crystallization (Smith et al., 1996). The crystals grown in space were larger and free of imperfections compared with crystals grown on Earth. Strikingly, whereas data to 1.9 Å resolution were obtained using the crystals grown on Earth and a laboratory X-ray source, data to 1.4 Å resolution were obtained using the space-grown crystals and the same X-ray source. In follow-up
work, preliminary results indicate that data to 0.9 Å resolution have now been obtained using synchrotron X-ray radiation on T6 insulin crystallized on the space shuttle in 1998 (G.D. Smith, personal communication). This ultrahigh resolution data is allowing very detailed analysis of the molecular structure, including the study of electronic distributions within the protein molecule.
The four cases described above provide the most convincing data currently available on the benefits of growing protein crystals in the microgravity environment. The 32 other experiments that produced space-grown crystals with improved resolution also support the potential value of microgravity. However, in some of these other cases, the investigators were not able to make the comparisons needed to demonstrate that growth in the microgravity environment was indeed the factor responsible for producing higher quality crystals. It is difficult to carry out a completely controlled experiment, particularly for cutting-edge projects that are intrinsically difficult and that involve structure determinations of immediate interest to researchers. It is not enough to compare space-grown crystals to crystals grown on Earth in the same equipment and solution over the same time period; the microgravity-grown crystals must also be compared to the best result from all Earth-based attempts at growing the crystal regardless of crystallization conditions, equipment, or time of growth. This latter comparison is the baseline standard for defining success.
The complexities that arise when trying to make appropriate comparisons are exemplified by in the case of the restriction endonuclease EcoRI complexed with DNA (EcoRI-DNA). High-quality crystals for this assembly have been obtained in microgravity (J. Rosenberg, 1999, submitted to Proteins), and the diffraction data obtained from the space-grown crystals are of significantly better quality than those obtained from similar samples on Earth. This is of considerable interest since the study of protein-DNA complexes is often plagued by crystals of poor quality. While this study demonstrates that biologically important results can be obtained from protein crystallization in space, it turns out that the incorporation of additional features in the analysis of the space-grown crystals, such as the use of cryogenic techniques and synchrotron radiation, makes it difficult to be certain that the improvements are due to microgravity and not to some of these additional factors (J. Rosenberg, personal communication). A direct quote from Dr. Rosenberg nicely summarizes the problem: “Significant improvement in the resolution of our EcoRI-DNA cocrystals . . . was due to a number of factors, including microgravity . . . [H]owever, I'm not in a position to untangle all the factors and just don't have the data to say how much of the improvement was due to each of the factors.”
Potential Areas of Future Impact
There is now a certain amount of evidence that crystal growth in a microgravity environment can have beneficial effects on the size and intrinsic order of macromolecular crystals. In many cases, crystals obtained in space are larger, have lower mosaicity, and diffract to higher resolution than comparable crystals grown on Earth. However, space-based crystallization programs have been very limited in scope in terms of the total throughput of samples compared with the enormous reach of modern protein crystallography on Earth. In addition, space-based crystallization efforts have been carried out under extremely adverse conditions. Therefore the results of the program, while intriguing, have had an extremely limited impact on biology during a time when technological innovations on the ground have produced significant and fundamental advances in our understanding of protein behavior and interactions.
However, despite the greatly increased sophistication of ground-based protein crystallization projects, the crystals of many important targets have suboptimal diffraction characteristics. Improvements in diffraction that move a system from the margins of structure determination (3.5 to 3.0 Å) to well beyond that boundary will have a significant impact on the ability of the resulting structure to provide important insights into biological mechanisms.
An example of such a situation is provided by the potassium channel. Potassium channels are integral membrane proteins that are important elements in the functioning of neuronal cells, and they also play diverse roles in the physiology of many different cell types. The potassium channels of greatest interest are those found in mammalian, particularly human, cells. However, it has not yet been possible to obtain crystals of mammalian potassium channels that are suitable for X-ray crystallographic analysis. Instead, crystals have been obtained of a
bacterial homolog that is similar in structure to the central core of mammalian potassium channels. While this analysis has provided a structural model for potassium channels and has revealed the general features of ion conductance and selectivity, diffraction data from these crystals are very weak and anisotropic at better than 3.5 Å. There would be enormous value in improving the structural accuracy of the model for potassium channels, but despite very significant efforts, better-diffracting crystals have not yet been obtained (R. MacKinnon, personal communication).
If the protein or proteins being crystallized are soluble, relatively stable, and well defined in their conformational state, there is little doubt that extensive experimental manipulation in the laboratory will eventually lead to better-diffracting crystals. For membrane proteins, such as the potassium channel, the difficulties appear to be much more serious. One reason for this is that the hydrophobic interactions that stabilize membrane proteins within the lipid bilayer are relatively nonspecific compared with the hydrogen bonding interactions that occur between surface side chains in soluble proteins. This makes it very difficult to obtain membrane protein crystals that diffract to high resolution, so membrane proteins are attractive targets for investigation in microgravity environments. Another general class of proteins yielding crystals that diffract very poorly are those that form transient complexes during dynamic events, such as during cellular signaling. There is great interest in obtaining high-resolution structural analyses of such protein complexes, and these may benefit from the particular conditions of microgravity. Drug design projects are another case where microgravity may be important. In the design of inhibitors it is usually important to see the stereochemistry by which binding occurs, and it is also necessary that the crystal structure be obtained for the precise target in question rather than for a closely related protein. This is a restriction that is usually avoided in practice, since the protein crystallographer will often search a set of closely related proteins for a protein with optimal crystallization characteristics. It is not at all uncommon to find that the particular protein that is most interesting, for example, the human variant of a family of proteins, does not yield suitable crystals.
The relatively poor diffraction obtained for such systems can arise for one or more reasons. These include the intrinsic flexibility of the macromolecular system being crystallized, as well as impurities or other factors that impede optimal crystal growth. At present there is no direct information on whether crystallization in a microgravity environment will have a positive impact in cases where the sole inhibitor of crystallization is the intrinsic flexibility of the molecules involved. Further experimentation will help resolve this question, but the controlled manner in which crystals grow in a microgravity environment may be beneficial in these cases.
Finding: While enormous strides have been made in protein crystallization in the last decade, it is still the case that there are very important classes of compelling biological problems where the difficulty of obtaining crystals that diffract to high resolution remains the chief barrier to structural analysis of the crystals. It is here that the NASA program must look to maximize its impact.
A prerequisite for all macromolecular structure studies is the availability of crystals that have suitable morphology and that are well ordered, sufficiently large, and stable enough to permit the recording of high-resolution diffraction data. Crystallographers will beat a path to any technology that can provide better quality crystals. Obtaining large crystals is probably not as important as it once was, because most crystallographers have access to very high brilliance X rays at synchrotron sources and can work with quite small samples. Similarly, cryogenic freezing techniques have been perfected to the point where most crystals can be kept stable indefinitely.
The main potential advantage of microgravity, therefore, is the possibility of obtaining crystals that diffract to higher resolution or crystals that have more favorable morphology. This could be especially important in structure-based drug design. Since the number of observable diffraction maxima increases as the inverse cube of the resolution, a crystal with a resolution of 2.0 Å yields nearly twice as many data as a crystal with a resolution of 2.5 Å. Determining the orientation of a small molecule inhibitor in an electron density map calculated from 2.5 Å data is problematic at best, whereas at 2.0 Å it will often show up quite clearly.
Potential Benefits of the Space Station Platform
Use of the ISS for future microgravity crystallization projects will probably lead to dramatic changes in the NASA macromolecular crystallography program. The length of experiments will increase significantly. Regular shuttle flights to and from the ISS will allow for considered planning of crystallization experiments. Improvement and standardization of the crystallization hardware will allow laboratory scientists to optimize crystallization procedures for the specialized hardware, maximizing the chances for success. The incorporation of microscopic examination on board the space station, a crucial element of successful crystallization in space, means that the crystallization process can be monitored and successful crystallization recognized when it occurs. The coupling of microscopic examination with automated procedures for crystal recovery and freezing will dramatically improve the ability of scientists to bring back high-quality crystals from space.
The benefits that structural biologists will realize as their use of the space shuttle on an ad hoc basis is replaced by the deployment of a dedicated protein crystal growth facility on the ISS can be seen as parallel to the benefits they realized when they started using synchrotron facilities dedicated to the production of X rays (see Box 1.1). For microgravity crystallization, the transition to a much more predictable and ordered regime on the ISS will have a maximum impact on modern biology if the projects chosen for experiments are ones that require improved crystallization to achieve significant scientific breakthroughs.
BOX 1.1 Analogy Between Synchrotrons and Space-based Research Platforms
There is a striking and instructive analogy between the development of synchrotron X-ray sources and the development of microgravity crystal growth facilities in the United States. Both rely on “big machines” and large capital expenditures of government money. The results of early experiments on both did not seem to many crystallographers to justify the expenditure.
First-generation synchrotrons, such as those at Cornell and Stanford Universities, were built for high-energy physics research. X rays were available for macromolecular diffraction studies only occasionally, and then only on a parasitic basis. While early experiments yielded some useful results, most investigators found these facilities difficult and frustrating to use. Often, the meager results obtained did not justify the time and money spent. In the 1970s and early 1980s, few of the early investigators could claim that synchrotron sources had had an important impact on structural biology. In 1982, the National Synchrotron Light Source, a second-generation machine dedicated to the production of X-rays, came on line at least 2 years behind schedule. Beam time on this machine was scarce, and most beam lines were not dedicated to macromolecular diffraction. However, early experiments soon proved the worth of synchrotron sources to all but the most skeptical.
This led to the development of third-generation machines such as the European Synchrotron Research Facility, the Advanced Photon Source, the Advanced Light Source, and Spring 8, with greatly increased X-ray brilliance, large numbers of user-friendly beam lines, and associated laboratories. These facilities have now become the X-ray sources of choice for most crystallographers.
By analogy, the space shuttle has been the first-generation microgravity platform. It was built as a space training and transport facility. Most microgravity experiments were consigned to small middeck lockers in the crew's living quarters. Generally, fewer than a hundred crystallizations per flight were attempted, and most were allowed to run for only a week or less. Although there have been some intriguing successes from the experiments carried out to date, at least as many crystals were lost before they could be returned to Earth-based laboratories for study. Few would claim that the program has yet had a significant impact on structural biology.
The International Space Station (ISS) may be considered the second-generation platform. Experiments will be carried out in dedicated racks in the ISS modules. Many more crystallizations will be set up and allowed to proceed for weeks or months, with periodic visual monitoring both on the ISS and from the ground. In addition, it may be possible to automate the process of crystal growth, monitoring, mounting, and freezing, and of obtaining diffraction data in microgravity owing to recent technological advances in hardware, especially the instrumentation being developed by scientists and engineers at the Center for Macromolecular Crystallography in Birmingham, Alabama.
It was the second generation of synchrotrons that demonstrated to the scientific community at large their potential value to structural biologists, and the ISS has the potential to produce the data necessary to resolve the worth of microgravity crystallization in a definitive fashion.
In order to engage the research community, NASA must focus its support on programs that are developing technologically innovative equipment and engaging in the structure determination of crystals with important biological implications. While past NASA-supported research on the crystallization process has not been without value, NASA's priority should now be to resolve questions about the usefulness of protein crystal growth in the microgravity environment to tackle important biological questions. Until the uncertainty about the value of space-based crystallization is resolved, a program of this fiscal magnitude is bound to engender resentment in the scientific community.
Potential for Interest from Commercial Entities
The focus of the task group's charge was to evaluate NASA's biotechnology program and facilities for the ISS. However, the task group also considered the related question of what role industry might play in developing what will be a large international facility for protein crystal growth. Commercial users of a macromolecular crystal growth facility on the ISS would come almost exclusively from pharmaceutical and biotechnology companies, with perhaps an occasional user from a contract research organization or an instrument manufacturer. Worldwide there are currently more than 70 companies with research programs in macromolecular crystallography. In aggregate these industrial organizations employ approximately 300 scientists and technicians with all levels of expertise in crystallography. Most are located in countries that already participate in development of the ISS.
All of these companies employ crystallographers to aid in the design of biologically active molecules for use in human and animal health care or agriculture for the production of food and fiber. Industrial research programs in macromolecular crystallography fall within two broad categories. In structure-based drug design, the three-dimensional structure of a target macromolecule is determined to help in the design of a compound, most often a small molecule, that will bind tightly and selectively to the target, modifying its activity. In macromolecular engineering, the structure of a macromolecule is determined in order to guide research aimed at changing its structure. The goal is to alter its properties in some desirable way, with the final commercial product being the mutant macromolecule itself.
Although many pharmaceutical and biotechnology companies have participated in the microgravity crystallization research, not one has yet committed substantial financial resources to the program. This is likely to remain the case until the benefits of microgravity can be convincingly documented by basic researchers and until facilities in space can handle greatly increased numbers of samples in a much more user friendly manner. The future financial participation of industry is, however, not out of the question. Again, the analogy of synchrotron beam lines is appropriate. About a decade ago, 12 of the largest pharmaceutical companies doing macromolecular structure research in the United States formed a consortium to build beam lines at the Advanced Photon Source. 2 To date these companies have invested approximately $10 million in what appears to be a very successful venture.
Goals and Potential Impacts of the NASA Cell Science Effort
The mission of the cell science program in NASA's Microgravity Research Division is to obtain new knowledge and increase the understanding of how low gravity influences fundamental cell biology with respect to tissue formation and space exploration. A variety of factors motivate this work. A primary goal is the need to understand the potential impact of the microgravity environment on the cell, tissue, and organ system functions of astronauts
More information about the Industrial Macromolecular Crystallography Association and its 12 member companies is available on the Web at <http://www.imca.aps.anl.gov/>.
spending months in space and on biologically based life-support systems such as those for plant growth or waste treatment. Cell science investigations on the ISS may help to foresee problems encountered by future longer-range space travelers. The research also has implications for ground-based systems, as perturbations of biological systems by microgravity can provide insight into physiological control in the absence of mechanical forces and in the absence of convection. This work could give scientists insight into how cellular processes respond to mechanical and chemical manipulation, eventually allowing them to design more efficient bioprocesses and to develop a new generation of high-resolution biosensors. Finally, the program also allows investigators to compare various tissue culturing techniques to determine which of them produce systems that most effectively mimic the characteristics of genuine tissue.
NASA's program on cell science in the microgravity environment is fairly young.3 As researchers gained experience the goals of the program broadened and the potential impact of the work became better understood. Originally, NASA focused on the generation of three-dimensional tissue constructs and on the rough characterization and comparison of these constructs to natural tissues, envisaging the commercial exploitation of space for generating large amounts of tissue. The task group does not believe such a goal is realistic and is encouraged to note that recent NASA-funded work has focused on more basic research. In the long-term, NASA' s work in cellular biotechnology is aimed at proof of concept and the development of a research platform for external investigators. According to NASA, its criterion for success is having the NASA-funded work of today lay the groundwork for a big breakthrough 10 years from now. In what area such a breakthrough will occur is not easy to predict, but a wide-ranging program on cell science in microgravity has the potential to impact a number of areas. In the broadest of terms, these areas include an increased understanding of the basic cell biology of terrestrial life in space and the effects of gravity on basic cell biology on Earth; the production of biopharmaceuticals and of functional tissue constructs for research and medicine; the propagation of organisms for antibiotic and vaccine development; and the identification of technologies that will result in new products to advance scientific capabilities on Earth. More specific areas of impact might include the use of bioreactors for efficient high-fidelity production of complex proteins requiring significant post-translational processing; the propagation of parasites for evaluation of function; the propagation of tumor tissue for evaluation of responses to therapeutic options; the miniaturization of analytical equipment such as flow cytometers, mass spectrometers, and sensing systems; a better understanding of gene expression within the three-dimensional context of cell and tissue architecture; and an appreciation for the consequences of modified gravitational forces during launch, sustained periods in space, and reentry. Because many aspects of cell behavior and tissue growth under low gravity conditions are not well understood, the basic cell science research carried out on the ISS will increase the amount of information available to the community. Whether this new knowledge about cells and tissues in different environments will fundamentally alter the scientific understanding of biological functionalities remains to be seen.
The enormous range of specific biological questions that fall within the general goals of the NASA cell science program is both an opportunity and a burden. On the one hand, since it is difficult to say which specific area has the greatest potential for a significant discovery, it could be a mistake to eliminate entire branches of research from contention for NASA funding. On the other hand, resources available on the ISS, such as equipment volume and crew time, will be limited, and in order to most effectively exploit this new research platform, NASA, in consultation with a committee representing the cell biology research community, might want to more narrowly define a subset of areas in which to support investigations. A clearer statement of goals would allow instrumentation developers to focus on specific needs for culture environments and analytical equipment on the ISS. Also, if focused on a few specific program areas, ground-based NASA-sponsored research projects might be more likely to construct the body of knowledge that would enable the experiments on the ISS to produce the future big breakthrough sought by NASA.
A small amount of funding (less than $1.5 million per year) was allotted to cellular biotechnology work within the Microgravity Research Division from 1983 to 1992, but significant resources (over $5 million per year) were not devoted to the program until the 1993 fiscal year.
Finding: It is appropriate for NASA to support a cell science program aimed at exploring the fundamental effects of the microgravity environment on biological systems at the cellular level. Results from such basic research experiments could have a significant impact on the fields of cell science and tissue engineering. However, the specific important questions within cell biology that can best be tackled on the ISS do not seem to have been defined yet. Narrowing the broad sweep of the current program might focus instrument development efforts and accelerate progress toward complete understanding of the effects of microgravity on specific biological phenomena.
The scope of NASA-supported cell science research is broadening. The research now goes beyond evaluating a limited number of structural and functional parameters and is taking a more mechanistic approach, using gene expression measurements, for example. It is also moving beyond a focus on instrument development to investigating the science that can be achieved using the new equipment. Detailed analyses at the biochemical and genetic level have demonstrated that microgravity has statistically significant effects on fundamental biological processes such as signal transduction and gene expression. Thus, microgravity, as an experimental parameter, may provide insight into fundamental aspects of biological regulation that will be important in terrestrial as well as extraterrestrial environments. Further, among systems tested to date, tissue constructs grown in microgravity have shown a unique utility for supporting studies on viral and pathogen culture. Key areas in which perturbations of cell structure and function in the extraterrestrial environment might be observed are components of nuclear architecture, cytoarchitecture, and the extracellular matrix. It is becoming increasingly evident that the organization of genes and regulatory proteins within the nucleus, the organization of nucleic acids and signaling proteins in the cytoplasm and cytoskeleton, and the organization of regulatory macromolecules within the extracellular matrix contribute to the physiologically responsive fidelity of gene expression. Consequently, the functional interrelationships between cell structure and gene expression within the three-dimensional context of cell and tissue organization should be rigorously and systematically studied under microgravity and regular Earth-gravity conditions. The corollary is that microgravity conditions can provide valuable insight into structure-function interrelationships that connect control of gene expression to cell and tissue architecture. Regulatory events within the three-dimensional context of cell and tissue organization should be further pursued under microgravity conditions and compared with similar constructs under controlled conditions. The information gained in these sorts of studies will certainly be useful, but it is important to note that, given the large number of signaling molecules and genes that could be investigated, a specific theme or focal point to the NASA program would improve the prospects of fully characterizing any particular cell structure-gene expression interrelationship.
To date, NASA's work in cell science has taken place on shuttle flights and on the Mir space station. These experiments, and investigations using other space facilities, have demonstrated that microgravity and the space environment affect cell shape, signal transduction, replication and proliferation, gene expression, apoptosis, and synthesis and orientation of intracellular and extracellular macromolecules (Dickson, 1991; Moore and Cogoli, 1996; Cogoli and Cogoli-Greuter, 1997; Lewis et al., 1997; Freed et al., 1997; Hammond et al., 1999). With the increased availability of research opportunities on the ISS and the new hardware developed specifically for this platform, further investigation of these processes may clarify how cells behave in the microgravity environment. For example, a deeper comprehension could be sought about the mechanisms behind the observed effects of microgravity on cells relevant to human physiology (e.g., muscle, bone, balance, circulation); understanding of how the cells in these systems sense and respond to mechanical stresses (i.e., the absence of gravitational acceleration associated with microgravity) would have immediate relevance for NASA's manned space program.
Potential research topics would not be limited to areas that have already been explored, but could come in other areas, including the adaptive responses of cells in microgravity to factors such as radiation; induced phenotypic and genotypic changes; selective pressure of the space environment on replicating cells; and the effect of microgravity on plant cells and tissues, on microorganisms (e.g., those that cause disease or that will be used for sewage treatment on long-range flights), and on cells (e.g., osteoblasts) that may not proliferate in bioreactors as they are currently designed. More general areas of study might include bioreporter models and sensors for biomolecular signatures and propagation of obligate and facultative parasites.
A key element in NASA's program in the future should be a concerted effort to understand the artifactual effects of the microgravity environment on cell science experiments (NRC, 1998). That cell and tissue constructs behave differently in space has been established; experiments designed to pinpoint the causes of the changes are the logical next step. In microgravity, the fluids that make up the culture environments behave quite differently than on Earth, and the differences in mass transport, convection, and buoyancy-driven flows could have a variety of effects on cells and tissue that are not directly related to the absence of gravity. While it may be reasonable to expect gravisensing cells to significantly alter their behavior in space, explaining the response of non-gravisensing tissue grown in the microgravity environment is unlikely to be straightforward. Efforts to separate the effects of low gravity on cells from the effects of low gravity on the cell growth medium will require meticulous experimental design, quantitative measures of cell alteration, and careful investigation of multiple experimental control groups. A thorough exploration of the factors affecting cell behavior in space will not only increase understanding of the effects of microgravity on cell and tissue formations but may also reveal unexpected information about the basic interactions between cells and their growth media.
Experimental Design and Instrumentation
The cell science program encompasses a wide range of research topics, from cancer cells to parasites, from chondrocytes to Bowhead whale cells. Many of the ongoing projects are thematically linked by a focus on three-dimensional tissue formation, and some success in this area has been achieved both in space and in ground-based experiments. However, tremendous progress has also been made in three-dimensional tissue development on Earth under unit gravity, using, for example, scaffolds and extracellular matrix gels. In experiments done in space, cell cultures experience a different gravitational environment, which reduces convection, buoyancy-driven flows, and sedimentation, and it is difficult to separate the various factors causing differences between space- and Earth-grown samples, making it difficult, in turn, to determine the appropriate experimental controls for space research. Possible approaches include the use of bioreactors on Earth, culture bags in the microgravity environment, bioreactors in space, three-dimensional structures grown on Earth from scaffolds, and the same experimental setup operated in the on-orbit 2.5-m centrifuge to restore the effects of unit gravity. The task group believes that for each experiment done in space, several potential control groups must be evaluated. Ordinary tissue culture flasks or spinner flasks on Earth are not appropriate benchmarks.
A related issue is the need to develop and apply quantitative measures for evaluating the results of cell science experiments performed on the ISS. In the space shuttle-based research to date, technical limitations, such as unstable environments and reentry effects, have produced experimental results that, while provocative, were essentially descriptive and phenomenological. Measurements are needed that can illuminate the molecular mechanisms underlying cellular functions. Work on techniques such as gene expression enables a more detailed accounting of what has been observed and can focus future research on areas where the effects of microgravity are greatest. Quantitative approaches may also help researchers to distinguish among the array of factors that may be influencing cell and tissue characteristics in the microgravitiy environment. Currently, researchers are limited by the difficulties inherent in distinguishing the effects of launch, flight, and, in many cases, reentry on samples. Experiments should be designed to separate these effects; such experiments, and the careful interpretation of the resulting data, will require close and frequent interactions with investigators in NASA's Life Sciences Division, as discussed below. A better understanding of these effects, as well as the development of quantitative approaches, will also assist in determining whether bioreactors, which were originally developed to simulate the microgravity environment, provide appropriate predictions of the behavior of cells and tissue in such an environment. The answer to this question will not be known until comparisons are made with experiments that have been subjected to microgravity environments and not modified by launch and reentry.
Finding: Appropriate experimental controls for space-based cell science experiments have not yet been determined. The best controls would be those that enable researchers to separate and investigate the multiple factors—including launch and reentry, effects of microgravity on the culture medium, and direct effects of microgravity on cellular behavior—that produce the changes observed in cells and tissues grown in space. Analytical techniques
that measure the molecular mechanisms underlying cellular functions will be essential to provide data for comparing proposed experimental controls and quantifying the observed changes in cell and tissue samples.
Requirements for Interprogrammatic Coordination Within NASA
At NASA, the work viewed by the task group was being carried out in the biotechnology section of the Microgravity Research Division. The themes of the cell science work under way in this program overlap with the scope of the work ongoing in the NASA Life Sciences Division, such as research on bone formation and muscle function. The complementary nature of these two programs needs to be recognized so that NASA personnel and external researchers can take full advantage of the resulting synergies. The Life Sciences Division covers a broad array of topics, including cellular and molecular biology, gravitational ecology, and organismal and comparative biology, that potentially relate to the cell science work under way in the Microgravity Research Division. Clearly, NASA and NASA-sponsored researchers would benefit from sharing and coordinating experiments on similar cell biology projects, such as the work on muscle growth and on osteoblasts. There is also a potential for synergy in connection with the Life Sciences Division's work on larger systems. Through this research, the observations of whole organisms provide a basis for theories about what happens to cells and tissues in microgravity, while the Microgravity Research Division's cell science program greatly expands the range of hypotheses that can be tested. Cooperation between the two programs provides a way to relate observations on cellular constructs to whole-animal response, for example, whether gene expression patterns in microgravity tissue constructs are similar to gene expression patterns in the corresponding animal organ in response to microgravity. If NASA-sponsored research is to provide a cellular basis for understanding the physiological effects of prolonged weightlessness on astronauts, a more complete continuum between the cellular experiments and whole organism responses needs to be established, and nonoverlapping but related contributions from both the Life Sciences and Microgravity Research Divisions are necessary.
Coordination (not just communication) between the divisions on cell science work is needed for fully understanding the relationships between in vivo and in vitro systems, and a better sharing of resources and expertise seems essential. While there is already overlap in flight hardware availability (both life science and microgravity researchers have and will continue to have access to the same equipment), it is not possible to have projects that are jointly funded by the Life Sciences Division and the cell science section of the Microgravity Research Division. The task group believes that although the potential synergies are significant, they are not yet being realized. A mechanism to establish cosponsored projects should be considered, possibly via joint NASA research announcements. It is important to note that each program does have unique approaches, goals, and perspectives, and to maintain these valuable differences, the administrative integrity of the separate programs should be retained.
Recommendation: The research strategies and projects of the cell science work in the biotechnology section of the Microgravity Research Division should be more closely coordinated with the work of NASA's Life Sciences Division to take advantage of overlapping work on bone and muscle constructs and of potential synergies between in vitro and in vivo research projects.