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--> 2 Cell Biology Introduction The discipline of cell biology examines biological processes at the level of the basic unit of biology, the cell. While increasingly incorporating a molecular viewpoint on the one hand and extending to the tissue level on the other, investigations in cell biology focus principally on events intrinsic to individual cells and on cellular responses to environmental factors. Cell biology therefore provides the underpinning for other disciplines relevant to space biology, including developmental biology; muscle, bone, and mineral metabolism; cardiopulmonary and other homeostatic systems; immunology; and sensorimotor integration (see individual chapters of this report). Each of these areas of inquiry at the tissue and organism levels ultimately depends on the normal function of individual cells and their integration into physiological networks. The Goldberg report1 devoted no specific chapter to the role of cell biology in space research, but instead incorporated cell biology within chapters on various physiological systems. That this report begins with a discussion of cell biology is a testament to the dramatic growth during the past decade in our understanding of fundamental biological processes at the cellular level and to the increasing applicability of cell biology to physiology at all levels. This growth in understanding has derived from a number of major technical developments and powerful new approaches to the study of the eukaryotic cell, among them the following: Molecular genetics and molecular biology, and their application to systems ranging from single cells to intact multicellular organisms .2 3 The introduction of new and improved techniques, including subtractive hybridization, differential display, and the polymerase chain reaction (PCR), have revolutionized the isolation and identification of both new genes and new members of known gene families. Similarly, approaches using both naturally occurring mutations and experimentally modified genomes (generated by [over]-expressing genetic constructs, including antisense or dominant-negative elements,
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--> or modification of the genome by homologous recombination [i.e., "knock-outs"]) have shed substantial light on many basic processes. Previously recognized cellular genes (e.g., growth factor receptors, adhesion molecules, signal transduction molecules) are being cloned and their regulation and gene products examined in increasing detail. In addition, the Human Genome Project and completion of the genome sequences of several model organisms are providing powerful tools. The increased recognition of the conservation of genetic homologies across evolution, and a heightened appreciation of the importance of such homologies in unraveling complex biological processes, emphasizes the significance of these projects. The ever-increasing sophistication of gene sequence databases, including those that consider not only primary sequence but also the predicted three-dimensional structure of the product proteins, provides new insights into protein function. Finally, there are ongoing advances in the design of reporter genes and sophisticated, sensitive detection apparatus for assessing transcriptional activity. Associated with this, detection hardware is being developed that can, in principle, be linked by telecommunications technology to remote sites for real-time analysis. Imaging technology.4 5 6 7 Confocal microscopy and real-time video microscopic methods (e.g., green fluorescent protein-tagged molecules), coupled with increasingly sophisticated computer-based image enhancement and processing techniques, are active areas of development, providing novel views of cellular processes, often visualized with the help of antibodies tagged with increasingly sensitive fluorochromes. Cell culture methodology. Growth factor biology, including the interactions of cells with extracellular matrix components, cell cycle regulation, and genetic mechanisms of programmed cell death, offer new insights into requirements for cell survival and differentiation. These advances allow researchers to study primary cultures of cells isolated directly from animal tissue, thereby alleviating the need to study transformed clonal cell lines. Progenitor cells of many cell types, including bone and muscle, can be grown in culture under conditions leading to their regulated differentiation.8 Dissociated cell cultures are increasingly being augmented by studies of microsphere, micromass, and bioreactor culture technology,9 as well as explant, slice, and reaggregating cultures, providing intermediate steps between dissociated cell populations and intact tissue. Recognition of marker molecules to identify sites of initial appearance, migration, and differentiation of specific progenitor cells allows analyses of lineage-specific events of cells both in culture and in vivo, including microenvironmental regulation of origins, migration, and differentiation. Questions regarding the extent to which necessary culture conditions can be maintained in the space environment, and thus the usefulness of culture techniques in the analysis of spaceflight biology, are considered below. Protein chemistry.10 11 12 Micro- and submicromethods for the detection and analysis of proteins have also advanced. These include immunochemical and immunohistological techniques, genetic modification of proteins to incorporate immunological tags or fluorescent markers, chemical crosslinking methods to detect and identify protein-protein interactions, and microsequencing to allow the synthesis of degenerate oligonucleotide probes for identifying and cloning the corresponding gene from genetic libraries. Macromolecular structure determination.13 14 Advances in instrumentation, including electron and x-ray diffraction, magnetic resonance, and mass spectrometry, coupled with dramatic developments in computer-based computational analysis and molecular modeling, have revolutionized structural biology. These results offer a new level of understanding about the way proteins function to produce specific physiological responses. As in other disciplines, this rapid and dramatic progress in what can be studied has meant that old questions can be asked in new ways, and asked at a level of experimental refinement that was previously
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--> unimaginable. In response to this expanded experimental capacity, a number of themes have emerged as major foci of cell biological research: How do cells replicate and maintain their genomes, including the regulation of their proliferative capacity and survival? These are questions that impinge not only on normal growth and development, including aging, but also on cancer biology. Identification of specific tumor suppressor pathways (e.g., p53), their interaction with genetically defined programs for regulating cell survival and death (e.g., ICE and bcl-2), and the elucidation of the regulation of telomere length and its influence on gene expression15 16 are a few examples. How do individual cells carry out genetically defined programs of differentiation and development into specialized tissues and multicellular organisms? Answers to these questions will help define the critical genetic and epigenetic events at the cellular level that lead to choices of specific lineage pathways in development.17 18 How do cells generate and maintain their complicated internal cytoarchitecture, including the cytoskeleton and the host of specialized membrane-bound organelles and membrane domains, thereby regulating both growth and form? This specific cellular substructure strictly defines both basic and differentiated cell function. Local perturbation at cell membrane focal adhesion complexes may cause profound effects to be telegraphed throughout the cell. This is done through the integration of individual components into a complex network coupling the cell surface to the cytoskeleton and to the nuclear matrix.19 How do cells synthesize and maintain organelle substructure? Differentiated function generally depends on specialized cell structure, as exemplified by such diverse cell types as myofibrils, neurons, polarized epithelia, and gravisensing cells in plants and the mammalian vestibular system. Our understanding of how cells address and target cellular material (including both proteins and RNAs) to produce and maintain the subcellular structure is also rapidly advancing, and specific molecular addresses and their receptors are being identified. Biophysical and biochemical principles underlying the assembly of multimolecular complex structures, regulation of assembly and disassembly, mechanisms for delivery of molecules to the specific cellular sites, and the role of the intracellular cytoskeleton are among the subjects being actively investigated.20 21 How do organisms respond at the cellular level to changes in their extracellular environments? This question integrates central, diverse concerns of cellular research. Cells interact with their environments through two major signal pathways: one based on soluble growth factors and their receptors (including those for hormones, growth factors, chemokines, and other small molecule effectors), and the second involving molecules responsible for direct interactions between cells (e.g., cadherins, selectins, CAMs)22 and extracellular matrix (integrins, collagens, glycosaminoglycans, laminin, fibronectin, and so on), including focal adhesion complexes. These two systems interact at the cell surface. The ligand-receptor interactions activate multiple interactive signaling complexes and pathways of intracellular signal transduction, leading to signal amplification.23 Finally, these metabolic cascades bring about changes in gene expression that govern cell physiology and behavior. Specific points of inquiry include mechanisms of reception or detection and response to extracellular chemical signals, environmental stress, and mechanical forces (e.g., touch and stretch-sensitive ion channels, fluid flow-shear forces, muscle and bone loading and unloading), including gravity, by mechanoreceptors. Previous Cell Biological Research In Space Studies of effects of the space environment on mammalian cells (in particular, cells growing in culture) began in the earliest days of spaceflight and have continued to the present. A number of
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--> compendia and summaries of these experiments have been published. For example, Dickson24 has presented a useful summary of all flight experiments using isolated cells (microbial and plant, as well as mammalian) through 1990, and Sahm and co-workers25 and Moore and Cogoli26 27 have summarized many studies conducted through 1994. In addition, data from IML-1, SL-J, and IML-228 are now becoming available. A broad range of effects of spaceflight on the cellular physiology of human lymphocytes, embryonic lung cell lines, and other cell types have been observed. These include changes in proliferation, genetic expression, signal transduction, morphology, motility, and cell-cell interaction and energy metabolism. A detailed evaluation of this extensive body of data is beyond the capacity of this report; however, several general problems are evident. Overall, the results have often proved inconsistent or contradictory. Many of the experiments have not been adequately controlled or replicated. Engineering restraints have often compromised experimental rigor. Unexpected technical difficulties have arisen. In many cases, the experimental findings have not been published in peer-reviewed journals. Fully evaluating the true physiological significance of many of the reported effects is often difficult. Moreover, our understanding of mechanisms responsible for these changes is still limited. In particular, which of these effects should be attributed to direct effects of gravity on cells, and in contrast, which are the result of indirect effects resulting from alterations of the cellular environment? In retrospect, it is likely that many of the described cellular responses of single cells to spaceflight (especially those growing in culture) can be attributed to indirect effects of microgravity and/or other aspects of the flight environment.29 30 The lack of sedimentation and thermal convection in microgravity is of particular concern because it results in the formation of stationary boundary layers around cells, seriously reducing nutrient uptake, gas exchange, and the removal of toxic products. The importance of providing adequate nutrient and gas exchange has been convincingly demonstrated by Musgrave and co-workers31 for intact Arabidopsis plants in-flight; similar considerations would surely hold for cells in culture. Decreased use of glucose noted in cells undergoing spaceflight compared with ground controls can also most likely be accounted for by limitations in the accessibility of nutrients and/or oxygen in the absence of convective flow, which in turn result in decreased cellular growth rates. An alternative hypothesis32 attributed decreased glucose utilization to a lack of a requirement for the maintenance of position of subcellular structures, or positional homeostasis. In addition, the induction of the cellular stress response by other perturbing factors in the environment (including strong hypergravity and vibration levels during launch and reentry) is undoubtedly important. Finally, the potential for enhanced radiation damage must be considered33 (see Chapter 11), although appropriate shielding and short-duration studies can minimize such effects. These and other problems are likely to be confounding variables that are difficult to evaluate by ground controls and that preclude unambiguous identification of microgravity per se as the agent responsible for a given effect. Nevertheless, the question of whether single cells might sense gravity directly and if so, how, has received theoretical consideration. Some researchers have proposed that single normal-sized cells (e.g., ˜10-μm diameter) simply do not weigh enough for gravitational forces to compete with much larger molecular forces.34 35 The question then is, Can forces acting on single cells be amplified to a physiologically significant signal? Examples have been proposed: Interaction of plasma membrane integrins with the extracellular matrix or substratum may transduce a reorganization of the intracellular cytoskeleton, with profound effects on cell behavior and gene expression;36 inherent amplification and adaptation (detection of relative changes, potentially over a wide range of input) of cellular biochemical networks are being analyzed in ever greater detail.37 Other hypotheses based on the nonlinearity of cellular molecular processes have suggested that single cells might amplify weak gravitational forces by
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--> virtue of nonlinear state transitions.38 The development of novel theoretical approaches to gravisensing by single cells could, in principle, lead to specific, testable hypotheses. In summary, experience from numerous previous studies, both in-flight and ground based, has highlighted certain pitfalls that can and must be avoided in the design and analysis of future experiments. Thus, the design of experiments in cell biology should evolve from the extensive and growing context of basic cell biological research. There is a strong current trend toward the investigation of cell biology at the level of molecular mechanism. Similarly, space cell biology should emphasize the identification of molecular mechanisms by which cells and tissues respond to spaceflight conditions. A few well-chosen model systems should be used to facilitate comparisons among experiments and create a reliable baseline of data. Experiments should begin with extensive ground-based analyses of normal (i.e., nonspaceflight-stressed) cell biology. Growing consideration should be given to studies on cells in situ, using steadily improving techniques for the analysis in intact tissues and organisms of cellular physiology from initial gene expression to cell architecture (e.g., in situ hybridization, single-cell PCR, confocal microscopy, patch-clamping, etc.) Finally, there have been difficulties in critically evaluating some studies in spaceflight that used cell culture techniques. To minimize these problems, experiments with cells in culture should be carefully evaluated before they are conducted, with thought given to their theoretical and practical justification, the availability of fully tested hardware, the capacity to carry out appropriate controls, adequate sample sizes, and the potential for repetition. The problem of the lack of sedimentation and fluid and gas convection in weightlessness must be considered. Differences in the fragility inherent among various cell strains and types also should be noted in choosing and comparing model systems. Generally, single cell culture models should be analyzed in ground-based studies. Opportunities For Nasa-Supported Research In Cell Biology As biomedical research as a whole moves toward the goal of understanding the molecular mechanisms underlying physiology, NASA-sponsored research over the next decade should focus on cellular and molecular mechanisms responsible for specific physiological phenomena in which microgravity or other stressful aspects of the space environment are significant. In other words, the space environment should be a potentially significant variable. For example, mechanisms by which cells respond to mechanical forces such as shear and gravity, and those related to environmental stress, are areas of special interest and opportunity for continued NASA emphasis. (See also the sections in this report on bone, muscle, cardiovascular physiology, immunology, and neuroendocrine and sensorimotor integration for additional focused discussions.) Mechanisms of Cellular Response to Mechanical Force Weight-bearing bone and skeletal muscle require the gravity-dependent mechanical force of compression on bone and contraction of muscle to maintain homeostasis of bone and muscle mass (see Chapters 6 and 7). Nevertheless, the molecular and cellular mechanisms whereby these tissues respond to modulate bone or muscle synthesis and resorption, turnover, and remodeling in response to gravitational forces are not well understood. These questions are central to space biology and medicine at the cellular level. Similar considerations hold for the investigation of other systems that respond directly to gravitational stimuli. These include the cellular mechanisms within the vestibular system involved in gravity reception and transduction of gravitational signals into perception of spatial orientation; gravitational
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--> response in plants (see Chapters 4 and 5); and activation or deactivation of touch receptors, stretch receptors, and other receptors sensitive to membrane distortion that are transduced into programmed changes in cell behavior and function. General mechanisms of mechanoreception and pathways of signal transduction from mechanical stresses are therefore recommended as areas of special opportunity and relevance for NASA life sciences. Many different cell types respond to various molecular and physical stimuli via a limited number of transduction pathways. Therefore, even though the final physiological effects may differ with stimulus and cell type, the probability is high that the tissue-specific response pathways to gravitational force will share common mechanistic features with other kinds of force-sensing pathways. Recommendation Studies of mechanisms of cellular mechanoreception should include identification of the cellular receptor, investigation of possible changes in membrane and cytoskeletal architecture, and analysis of pathways of response, including signal transduction and resolution in time and space of possible ion transients. Cellular Response to Environmental Stress Space is a stressful environment. Organisms have developed highly sophisticated mechanisms for coping with stress, not only at the organismic and physiological levels but also at the level of single cells. The mechanisms by which cells perceive, respond, and adapt to various kinds of environmental stress are a major research emphasis in cell biology and are relevant to understanding the mechanisms of response to the space environment.39 40 Recommendation Studies of cellular responses to environmental stresses encountered in spaceflight (e.g., anoxia, temperature shock, vibration) should include investigation of the nature of cellular receptors, signal transduction pathways, changes in gene expression, and identification and structure and function analysis of stress proteins that mediate the response. Development of Advanced Instrumentation and Methodologies Conducting sophisticated cell biology experiments in space successfully will require the development of highly automated and miniaturized instrumentation and advanced methodologies, many of which will be equally useful for ground-based research. The recommendations of a previous report that "dedicated microprocessors should be used for process control, data storage, or both, and rapid communication in real time with ground-based teams should be a goal" remain valid. 41 This goal can be realized in the current research climate and is being pursued with significant success.42 43 Recommendation NASA should work with the scientific community and industry to foster development of advanced instrumentation and methodologies for space-based studies at the cellular level.
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