This is the fifth post in our series The Impact of Gravity on Life, a paper written by Dr. Emily R. Morey-Holton, NASA Ames Research Center, Moffett Field, California. Read the series in its entirety here in blog posts tagged Impact of Gravity on Life.
Physics predicts that altered gravity will not cause any changes in cells because gravity is extremely weak compared with other physical forces acting on or within cells (Brown, 1991). Yet, cellular changes have been reported. Are the physical scientists wrong or are there other previously unconsidered factors at work on cells when gravity changes? Purely physical mechanisms for gravitational responses probably can be eliminated (Hemmersbach et al., 1999). Yet, cells appear to respond to changes in the environment (Klaus et al., 1997) and to have evolved structures that interact directly with the outside environment to sense the environmental loads placed upon them (Hemmersbach et al., 1999; Ingber, 1998).
The bacterium E. coli has flown experimentally in culture seven times aboard the space shuttle (Klaus et al., 1997). During spaceflight, E. coli exhibited a shortened lag phase, an increased duration of exponential growth, and an approximate doubling of final cell population density compared to ground controls. These differences may be related to the lack of convective fluid mixing and sedimentation, processes that require gravity. During exponential growth in minimal gravity, the more uniform distribution of suspended cells may initially increase nutrient availability compared to the 1-G- sedimenting cells that concentrate on the container bottom away from available nutrients remaining in solution. Also, local toxic by-products could become concentrated on the bottom of the 1-G container with cells in increased proximity to each other. Such a process could limit cell growth. Thus, changes in E. coli and possibly other cells during spaceflight may be related to alterations in the microenvironment surrounding non-motile cells. If true, then the extracellular environment plays a critical role in evolution of single cells through controlling nutrients and waste. This response to the extracellular environment suggests that intracellular gravity sensors are not essential for cells to elicit a gravitational response. Earlier predictions that microgravity could not affect cells were focused on the physical inability of gravity, an extremely weak intracellular force, to elicit an immediate or “direct” response from organisms of such small mass. Rather than a “direct” response, reduced gravity more likely initiates a cascade of events — the altered physical force leads to an altered chemical environment, which in turn gives rise to an altered physiological response. Modeling cell behavior predicts how cells evolve in different physical environments including Earth by including gravity as an integral part of the equations; hence, changes in sedimentation, convection, nutrient availability, and waste removal with altered gravity can be predicted.
Hammond, Kaysen, and colleagues (Kaysen et al., 1999) cultured renal cells under different conditions. They concluded that differentiation of renal cells in culture most likely requires three simultaneous conditions: low shear and low turbulence, three-dimensional configuration of the cell mass (i.e., free-floating), and co-spatial arrangement of different cell types and substrates. They have cultured human renal cells in rotating-wall vessels and in centrifuged bags on Earth, and in stationary bags flown aboard the shuttle (Hammond et al., 1999). Controls for all experiments were simultaneous, ground- based, bag cultures. All cultures contained liquid medium and the bags were made of material that was non-adherent for cells. A plethora of changes in steady-state level of mRNA expression occurred in space-flown human cells (1632 of 10,000 genes or 16.3%) compared to the Earth-based bag cultures. These patterns were unrelated to the changes in gene-expression found in rotating-wall vessel experiments. Shear stress response elements and genes for heat shock proteins showed no change in steady-state gene expression in the flight culture. Specific transcription factors underwent large changes during flight (full data set at http://www.tmc.tulane.edu/astrobiology/microarray). In the rotating-wall vessel, 914 genes or 9% changed expression. In the centrifuge, increasing gravity to 3-G caused only 4 genes to change expression greater than 3-fold. In addition to the unique changes in gene expression noted during flight, structural changes in the cultured rat kidney cells also occurred. Far more microvilli were formed in renal cells grown in space or in the rotating-wall vessel than in the 1-G static bag culture or during centrifugation (Hammond et al., 2000). These studies suggest that renal cells flown in space have unique patterns of gene expression unrelated to the best Earth-based model of spaceflight (i.e., rotating-wall vessel), and that the ability to form a three-dimensional, free-floating structure in culture appears critical to induce tissue-specific, differentiated features in renal cells.
The data from bacterial and renal cells suggest that spaceflight may affect cells via their external environment and that differentiation of renal tissue may be enhanced during spaceflight. Such studies are demonstrating how physical factors, specifically gravity, regulate expression of specific genes, creating an organism specific for that environment. Thus, some cells and tissue may show greater differentiation of specific features while other may show the reverse. In fact, the timing of gene expression may be beneficial or detrimental to downstream effects and, hence, alter the final protein product and, ultimately, the organism. Evolution is more likely to cause changes through altered gene expression rather than through genomic modifications as the latter are more likely spontaneous mutations. Data are indicating that gravity may actually be a critical environmental factor in determining the differentiation and maturation of cells on Earth.
Early results with cultured cells from the musculoskeletal system suggest that spaceflight induces a variety of responses. Delayed differentiation and changes in the cytoskeleton, nuclear morphology, and gene expression have been reported for bone cells (Hughes-Fulford and Lewis, 1996; Landis et al., 1999). Dr. Herman Vandenburgh has flown fused myoblasts (i.e., muscle fibers) to investigate the effects of microgravity on cultured muscle fibers. He found that flight muscle organoids were 10-20% thinner (i.e., atrophied) compared with ground controls due to decreases in protein synthesis rather than increases in protein degradation (Vandenburgh, 1999). Interestingly, atrophy of the isolated muscle fibers in culture was very similar to the amount of muscle atrophy reported in flight animals. These preliminary data from bone and muscle cells suggest that spaceflight affects adherent cells and tissues even when isolated from systemic factors and that the physical environment might direct the ultimate development of cells, organs, and tissues.
Changes in the physical environment surrounding cells, in vivo or in vitro, can lead indirectly to changes within the cell. Little is known about if or how individual cells sense mechanical signals or how they transduce those signals into a biochemical response. A cellular mechanosensing system might initiate changes in numerous signaling pathways. Such a system has been found in cells that attach to an extracellular matrix (i.e., the cell substratum) and the cellular components are beginning to be defined. These cellular interactions likely suppress or amplify signals generated by gravitational loading. We now know that the extracellular matrix to which cells attach contains adhesive proteins that bind to regulatory proteins that traverse the cell membrane. These transmembrane regulatory proteins (e.g., integrins), in turn, connect to the cytoskeleton and the cytoskeleton ultimately connects to the cell nucleus. Given these connections, activation of the regulatory proteins in the cell membrane can lead directly to regulation of gene expression, thereby eliminating the need for a solely intracellular gravity sensor. Living cells may be hard-wired to respond immediately to external mechanical stresses. Exciting research on the interaction of the cell cytoskeleton with membrane components and the extracellular matrix is shedding light on possible “force sensors” at the cellular level that might be essential for the differentiation process (Ingber 1997, 1998, Globus et al. 1998, Schwuchow and Sack 1994, Wayne et al. 1992). Ingber has applied to cells the concept of “tensegrity” (i.e., tensional integrity), a tension-dependent form of cellular architecture that organizes the cytoskeleton and stabilizes cellular form (Ingber, 1999). This architecture may be the cellular system that initiates a response to mechanical loading as a result of stress-dependent changes in structure and may have been a key factor in the origin of cellular life (Ingber, 2000).
Definition of the cellular connections that might sense and transduce mechanical signals into a biochemical response may also shed light on the events initiating cell maturation. As a cell matures, it stops dividing and begins to express characteristics of a mature cell type. If a cell does not mature, it will continue to divide–the definition of a cancer cell. The maturation process may be triggered by multiple factors, including loads placed on the extracellular matrix during different phases of development.
With exciting new molecular tools in hand and the development of facilities for increasing gravity on Earth (i.e., centrifugation) or decreasing gravity on space platforms, great strides will be made in understanding the influence of gravity in living systems at the cellular level within the next decade.
In summary, the local environment around cells may be altered in space. Such changes may affect cellular metabolism and steady-state gene expression may change. Potential adaptive systems in eukaryotic cells include force coupling through the cellular skeleton, ion channels, and other load-sensitive cellular structures that might alter cellular signaling. Further investigation into cellular changes at multiple gravity levels is required. Research may show that cellular architecture in eukaryotic cells evolved to oppose loading or amplify directional cues. Thus, physical changes in the aqueous medium surrounding cells in culture and cellular structures that oppose or respond to mechanical loads may provide cells with the ability to respond to gravity.