THE IN SPACEFLOWN CELLS: AN OVERVIEW Marian L. Lewis Department of Biological Sciences, University of Alabama in Huntsville

ABSTRACT Zond 5 and 6 spacecraft in the 1960s, describe cellular responses (Cogoli and Gmunder, 1991, Cogoli, 1996, Cytoskeletal changes in spaceflown cells include altered Cogoli and Cogoli-Greuter, 1997). From the 1980s to the and polymerization; morphological anomalies in actin present, many spaceflight investigations have sought to stress fibers, , and networks; discover why T lymphocytes are growth arrested in coalesced, shortened microtubules; loss of - microgravity. Spaceflight-induced changes to the membrane contact; perinuclear network loosening; cytoskeleton have long been implicated. Though still not disruption of microtubule organizing centers; and proven, accumulating evidence strengthens this view. complex anomalies during cell division. Cellular function depends on interaction of actin stress fibers, microtubules and Observed responses in spaceflown cells such as intermediate filaments with complex assemblies of hundreds of reduced secretory processes (Hymer et al., 1985, cytoskeletal accessory binding . Thus, spaceflight- Grindeland et al., 1990), blunted T lymphocyte activation induced cytoskeletal changes may affect cell shape, mechanical (Cogoli et al., 1984, 1991, 1993, Cogoli, 1996, Cogoli and support, cell movement, transport and position of RNA, Cogoli-Greuter, 1997), T lymphocyte (Jurkat) growth kinase C (PKC) and other cellular proteins, arrest (Lewis et al., 1998), altered PKC translocation segregation during mitosis, and in plants, cytoplasmic streaming (Schmitt et al., 1996, Hatton, et al., 1999) and changes in and polarized growth and orientation regulated by actin binding production (Grove et al., 1995) may result from proteins. In lymphocytes, microtubules disrupted by launch vibration appear to reorganize in microgravity but may not be anomalies in cytoskeletal structure and function. Recent typical. Cells adapt to mechanical stimuli by modifying reports describing abnormalities in cytoskeletal cytoskeletal structure and regulating signal transduction through morphology (Lewis et al., 1998) and altered cytoskeletal membrane inositol phospholipid interactions with cytoskeletal gene expression in spaceflown cells (Hammond et al., proteins such as . Gelsolin links cytoskeletal dynamics 1999, 2000, Lewis et al., 2001) further implicate aberrant (actin polymerization) to signal transduction at the cell inner cytoskeletal function in microgravity. membrane. We found gelsolin precursor message down- The microtubule cytoskeleton, disrupted in spaceflown regulation in cDNA microarray assays of lymphocytes flown on Jurkat cells and cells subjected to simulated shuttle launch the space shuttle. Of over 20,000 genes evaluated, eleven vibration, appears to reorganize within 24 hours (Lewis et cytoskeleton-related genes were expressed differently in flown compared to ground controls. I postulate that signal transduction al., 1998). However, cells in microgravity did not at the inner membrane, through the interaction of actin proliferate; whereas ground-based vibrated cells resumed polymerizing proteins and inositol phosphate hydrolysis growth. Using in vitro solutions of microtubules, Papaseit mediated by cytoskeleton binding proteins, is a mechanism for et al., (2000) and Tabony et al., (2001) found that self- reduced cell growth in space. organization of microtubules (not to be confused with addition and losing of tubulin subunits to microtubule INTRODUCTION filaments, or ) is dependent on gravity. Whether the putative reorganization in microgravity This review describes morphological and gene expression produces a functional cytoskeleton remains to be changes observed in the cytoskeleton of cells flown on the determined. The effect of simulated shuttle launch space shuttle (days), on sounding rockets (six to twelve vibration on cytoskeletal morphology (Lewis et al., 1998 minutes), and in ground-based clinorotated cells and cells and Lewis, 1999) and altered gene expression in Jurkat subjected to simulated shuttle launch vibration. The goal cells (a leukemic T cell line) provides evidence that is to update and summarize effects of spaceflight on the vibration alone does not account for reduced cell growth cytoskeleton and to provide information that may be response in T cells (Lewis et al., 2001). useful for interpreting changes observed in cells and Changes in spaceflown compared to ground control organisms, as well as humans and animals in altered cells are often described for established cell lines such as gravity environments Over the past forty years, a Jurkat, 3T3 mouse osteoblasts or other continuous culture number of spaceflight experiments have confirmed cell- cell types. Cell lines provide a readily available source level changes. The pioneering work of Konstantinova et and reduce variability characteristic of freshly drawn al., 1973 and Cogoli et al., 1984 and 1991 identified T lymphocytes. Frozen stocks make it possible to conduct lymphocyte sensitivity to microgravity. Reviews, repeated tests on cells of the same, or near-same, passage beginning with the Russian level. Jurkat cells have been flown in a number of spaceflight experiments (Limouse et al., 1991, Schmitt et ______al., 1996, Cogoli-Greuter et al., 1997a,b, 1998, Lewis et * Correspondence to: Marian L. Lewis al., 1998 and 2001, Sciola et al., 1999, Cubano and Lewis, Department of Biological Sciences, University of Alabama 2000 and 2001). Jurkat cells are easily and consistently 250 Hartside Rd., Owens Cross Rds., AL 35763 cultured and may be held in a quiescent state for several Email: [email protected] days at 20oC in medium containing 2% serum. Thus cells Phone: 256-539-3604 in microgravity may be growth stimulated simply by

Gravitational and Space Biology Bulletin 17(2) June 2004 1

M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview increasing serum from 2% to 10% and raising the by immunofluorescence microscopy for cells fixed in temperature from 20oC to 37oC. microgravity with formalin or formalin mixed with glutaraldehyde. After landing, cells may be stained with Cytoskeletal Function and Components rhodamine phalloidin, which binds directly to actin, or An intact, functional cytoskeleton is required during reacted with specific antibodies tagged with fluorescent mitosis to segregate and, in non-dividing antibody to identify other proteins. For instance, cells for cells, the cytoskeleton provides mechanical support, visualization of microtubules (MT) and intermediate maintains cell shape and spatially positions and filaments (IF) or other cytoskeletal proteins are cellular proteins. The cytoskeleton facilitates cell motility permeabilized with Triton-X 100 and reacted with the and movement of organelles and proteins within the desired specific antibody. Incubation with a second . During signal transduction, cytoskeletal antibody reactive to the first and tagged with a fluorescent elements form focal contacts that relay signals from dye such as fluorescein isothiocyanate (FITC), allows membrane receptors to the nucleus to initiate growth or visualization by use of a fluorescence microscope often other responses. All of these functions depend on the combined with confocal or electron microscopy. binding of cytoskeletal filaments with cooperating Techniques to evaluate gene expression include the complexes of proteins. Disruption of the cytoskeleton reverse transcriptase polymerase chain reaction (RT- compromises cellular function, growth and metabolism PCR), competitive RT-PCR, the RNase protection assay, and can ultimately lead to cell death. and cDNA microarray analyses. The expression of Three types of filaments (microtubules, actin stress thousands of genes in very small samples of RNA is fibers and intermediate filaments), interconnected and easily evaluated by cDNA microarray. Total RNA in coordinated by hundreds of associated cytoskeletal cells sampled in microgravity at desired times can be accessory proteins, make up the eukaryotic cytoskeleton. stabilized by adding a guanidinium solution or by freezing Binding in cooperative groups to cytoskeletal filaments, and RNA message can then be analyzed after landing. these proteins facilitate dynamic and rapid polymerization and depolymerization of filaments. Because binding can RESULTS AND DISCUSSION occur without producing measurable chemical changes, it Studies in clinostats is difficult to assess the cytoskeleton's role, at the The references for cytoskeletal changes found in ground- molecular level, in cellular responses in microgravity. based, altered gravity studies are shown in Table 2. Table 1 shows cytoskeletal-associated proteins. Using a fast rotating clinostat, Rijken et al., (1989) found increased cytoskeleton-related cell rounding in A-431 epidermoid cells cultured in the presence of EGF. Hoeger and Gruener (1990) showed that microtubules in Xenopus myocytes in a slow rotating clinostat became disorganized, condensed and associated in irregular patterns. These early experiments demonstrated that the cytoskeleton is sensitive to changes in gravity. Sarkar et al., (2000) subjected osteoblast-like ROS 17/2.8 cells to clinostatic rotation at 50 rpm and found that 35% of the cells detached from the substrate within two hours. Apoptosis, observed in many cells, appeared to be associated with disorganization of the actin cytoskeleton and perinuclear distribution of integrin beta 1. Apparently the change from unit gravity triggered apoptosis by modifying the organization of the cytoskeleton.

General Methodology Detailed methods are described by Lewis et al. for evaluating growth, glucose metabolism, apoptosis, and cytoskeletal morphology (1998) and gene expression (2001). In general, cytoskeletal morphology is evaluated 2 Gravitational and Space Biology Bulletin 17(2) June 2004

M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview Cytoskeletal changes during short-term low-gravity investigators suggest that the manner in which actin exposure are remodeled may affect signal Table 3 lists cell types or in vitro solutions subjected to transduction. In Jurkat cells flown on MAXUS and short-term low-gravity on parabola-flying aircraft and MASER sounding rockets, Cogoli-Greuter et al. (1997a,b, sounding rockets. Two studies, one flown on the KC-135 1998) and Sciola et al. (1999) found significantly aircraft providing repeated short-term, low-g of increased numbers of large bundles of in flown approximately 30 seconds and the other on a MAXUS cells compared to controls as shown in Figure 1k and l sounding rocket (13 minutes of low-g), showed changes Vimentin filaments were discontinuous (possibly in microtubule assembly in cell-free solutions of tubulin. depolymerized) after 30 seconds in microgravity. Moos et al. (1988) flew cell-free solutions of purified tubulin on the KC-135 aircraft and found differences in microtubule assembly between the low-g and 2-g phases of parabolic flight. Twelve years later in studies flown on the MAXUS sounding rocket, Papaseit et al. (2000) and Tabony et al. (2001), using cell-free solutions of purified bovine tubulin and GTP under permissive temperature, showed that microtubule self-organization does not occur if gravity is absent during the first 13 minutes.

Figure 1. Cytoskeletal abnormalities in spaceflown cells. Cells in microgravity (a, c, e, g, i, k) and controls (b, d, f, h, j, l). Actin filaments in Xenopus embryonic muscle cells flown on STS-52 and STS-56 (a), Guignandon et al. (1997) found that transitions compared to ground controls (b), showed increased cabling and through 1g, 2g, and micro-g during 15 to 30 parabolas decreased filament linearity (Gruener et al., 1994). The actin cytoskeleton in mouse MC3TC-E1 osteoblasts flown on STS-56 (c) had resulted in decreased cell area and focal contact plaque fewer actin stress fibers than ground controls (d) and was coalesced reorganization in osteosarcoma ROS 17/2.8 cells. These (Hughes-Fulford and Lewis, 1996). Microtubules in HL60 human experiments, continued on Russian Foton space flights myeloid leukemia cells flown on STS-69 (e) did not polymerize and (Table 4), confirmed that anchorage-dependent cells are appeared as globular dots, whereas microtubules in ground controls (f) initiated polymerization from MTOCs (Piepmeier et al., 1997). The sensitive to gravity transition and adaptation to changes in microtubule cytoskeleton in Jurkat flown on STS-76 (g) appeared to be gravity appear to involve microtubules. coalesced, shortened, arranged randomly and MTOCs were Boonstra (1999) summarized a number of studies disorganized compared to ground controls (h) (Lewis et al., 1998). showing that EGF-induced cytoskeletal changes and the Alpha-tubulin in cells of the human breast cancer line, MCF-7, flown on the orbiting Foton capsule (i) and fixed after 48 hours in microgravity, actin system are sensitive to changes in did not reorganize but appeared as tubulin subunits or very short gravity. Experiments in clinostats and centrifuges (Rijken microtubules. The in-flight 1g controls (j) formed a normal cytoskeleton et al., 1989) were confirmed by the sounding rocket (Vassy et al., 2001). Intermediate filaments of vimentin in spaceflown experiments showing that altered gravity increases the F- Jurkat cells (k) fixed after 30 seconds in low gravity showed bundling of filaments compared to ground controls (l) (Cogoli-Greuter et al., 1998). actin content in A431 epidermoid carcinoma cells. These (Bars, 5 µm.)

Gravitational and Space Biology Bulletin 17(2) June 2004 3 M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview The structure of actin on the inner side of the cell Piepmeier et al. (1997) reported that microtubules in membrane after Con A binding and co-localization of human myeloid HL-60 cells, in the presence of taxol, actin with Con A receptors did not appear to be changed. flown on the shuttle did not polymerize. Instead they Cogoli-Greuter ruled out cytoskeletal damage due to appeared as amorphic globular stained bodies of acceleration at 13 to 15g during rocket launches and depolymerized tubulin or very short of tubulin concluded that cytoskeletal anomalies were a direct effect as shown in (Figure 1e and f). In the ground controls, of microgravity on the cytoskeleton. The MASER flights under the same conditions of time, temperature, and also found that patching and capping seemed to be slower hardware, microtubules polymerization was evident. in flown compared to ground controls. However, Con-A In mouse 3T3-E1 osteoblasts flown on STS-56, binding to cell membranes appeared to be unaffected in Hughes-Fulford and Lewis (1996) found that actin microgravity. Cells appeared to be motile, cell-cell cytoskeletal morphology was significantly altered contacts occurred and membrane changes were not noted compared to ground controls in the same hardware and in the Jurkat cells. Primary human skin , held at the same temperature as the flight experiments exposed to six minutes of microgravity on a MASER (Figure. 1c and d). In flown cells, actin stress fibers were sounding rocket, showed no apparent changes in the altered. This may result from decreased integrin-related cortical actin cytoskeleton after electrofusion. And, attachment. The cytoskeletal morphology was also altered within the limits of the experiment, there was no effect on and cell growth in microgravity was slower than in cell fusion (Jongkind et al., 1996). ground controls. In the Biorack series, flown on STS-76, STS-81 and STS-84, Hughes-Fulford and Gilbertson Cytoskeletal changes in cells flown on orbiting (1999) also found elongation of the actin cytoskeleton and Spacecraft reduced actin cytoskeleton surface area compared to in- Experiments flown on the space shuttle and in the Foton flight 1g and ground controls. In addition, nuclei were orbiting capsule are shown in Table 4. Gruener et al. smaller, oblong in shape, and fewer punctuate areas were (1994) confirmed cytoskeletal sensitivity to altered evident compared to controls. gravity previously demonstrated by clinostat experiments In Jurkat cells fixed 20 hours after launch and four (Hoeger and Gruener, 1990) in experiments flown on hours after growth stimulation in microgravity by STS-52 and STS-56. Filaments in Xenopus embryonic increasing serum to 10% and temperature to 20oC on muscle cells appeared to be thickened and filament STS-76 (Lewis et al., 1998 and Lewis, 1999), the linearity was decreased (Figure 1a and b). The changes in microtubule cytoskeleton in many flown cells was distribution of actin appeared to be associated with coalesced and prematurely terminated and the reduced acetylcholine receptor aggregation at focal microtubule organizing centers (MTOCs) were disrupted contacts with polystyrene beads. (Figure 1g and h). The microtubule cytoskeleton in ground control cells radiated toward the from well-formed MTOCs. In contrast to experiments of Vassy et al. (2001) with human breast cancer cells flown on the Foton orbiting spacecraft in which the cytoskeletal elements did not reorganize, Lewis et al. found that the microtubule cytoskeleton appeared to re-form in microgravity. Based on the findings of others (Moos et al., 1988, Boonstra et al., 1999, Papaseit et al., 2000, and Tabony et al., 2001, 2002), the cytoskeleton though apparently reorganized, may not be properly associated in the absence of gravity. In addition to disorganization of MTOCs reported by Lewis et al. in spaceflown Jurkat cells, Schatten et al. (1999) also found disrupted MTOCs. In an experiment flown on STS-77, she showed that 4% of the dividing cells in sea urchin embryos had abnormalities in the -centrosome region. Further evidence of MTOC damage was provided by Lewis, et al., 2001, who found that message was up-regulated for a centriole-related protein (C-NAP1) in Jurkat cells flown on the shuttle. Cells subjected to simulated shuttle launch vibration also up-regulated C-NAP1. Anomalies in MTOC structure and genes regulating MTOCs could play a role in reduced cell division in microgravity. In human breast cancer MCF-7 cells flown in a Russian Photon orbiting space capsule, Vassy et al. (2001) reported that only a few microtubule filaments were apparent in cells sampled at 1.5 hours yet the 1g controls had numerous well- 4 Gravitational and Space Biology Bulletin 17(2) June 2004

M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview polymerized filaments (Figure 1i and j). By 48 hours, the Cytoskeletal gene expression in spaceflown T microtubule cytoskeleton in the 1g controls had lymphocytes (Jurkat) reorganized, however; the microtubules in cells in A summary of gene expression changes in a number of microgravity did not reorganize. The microtubules in cell types clinorotated, centrifuged, and vibrated in many of the microgravity cells still showed abnormalities ground-based studies compared to cells flown on the at 48 hours. The perinuclear cytokeratin network was shuttle and sounding rockets was published recently by loosely woven and numerous, very short microtubules or Lewis (2002). Only the expression of cytoskeleton- labeled free tubulin subunits were evident. Other related genes is described herein. anomalies included prolonged mitosis, reduced cell Lewis et al. (2001) evaluated RNA message by cDNA growth and a block the G2M phase of the cell cycle (also microarray for 4,324 genes at 24 hours and more than noted in Jurkat cells, Lewis et al. 1998). The ends of 20,000 genes and expressed sequence tags (EST) at 48 stress fibers at the cell periphery showed an apparent, hours in Jurkat cells flown on STS-95. Only two percent though not quantitatively analyzed, reduction in of the genes expressed in flown Jurkat cells were either phosphotyrosine labeling. The organization if up- or down-regulated by 2-fold or greater compared to microfilaments into stress fibers and signal transduction the ground controls. Message for ten cytoskeletal genes from focal contacts appeared to be decreased compared to was up-regulated and one was down-regulated compared 1g in-flight and ground controls. to ground control cells. These are shown in Table 5. The work of Guignandon et al. (2001) with ROS 17/2.8 osteoblasts flown on the orbiting Russian Photon capsule confirmed previously observed sensitivity to gravity change noted during parabolic flight by Guignandon et al. (1997). During Foton flight, was disorganized. This was associated with disassembly of vinculin and phosphorylated proteins in focal contacts. F-actin networks were found in the cortical area of cells. This research indicates that integrin-mediated is significantly affected in microgravity in these cells.

Effect of ground-based simulated shuttle launch vibration on the cytoskeleton

Jurkat cells were subjected to simulated STS-95 shuttle launch vibration at the NASA Marshal Space Flight Center vibration facility in Huntsville, Alabama. Expression differences were found for genes that Syringes containing cells suspended in culture medium encode metabolism, signal transduction, adhesion, were bolted to tables and vibrated in the X, Y, and Z transcription, apoptosis, and tumor suppression in planes. Effects on the cytoskeleton were similar to those addition to the cytoskeleton-related genes. The observed in spaceflown Jurkat cells (Lewis et al., 1998 spaceflight and ground-based vibration study results, and Lewis, 1999) as well as in other spaceflown cells. including 53 pages of gene expression data, may be Anomalies included disruption of the MTOCs (Schatten obtained as a PDF full text online publication (Reference: et al., 1999), filament bundling (Gruener et al., 1994, Lewis et al., FASEB J (June 18, 2001) 10.1096/fj.00- Hughes-Fulford and Lewis, 1996, Cogoli-Greuter et al., 0820fje). (The publication and tables of gene expression 1997a,b, 1998, Sciola et al., 1999) loss of filament data, listed as Space Genes, may be directly accessed by alignment and prematurely terminated cytoskeletal use of the URL:

filaments (Piepmeier et al., 1997, Vassy et al., 2001). http://www.fasebj.org/cgi/content/abstract/00- The cytoskeleton in flown Jurkat cells, sampled at 48 0820fjev1?ijkey=4kpP6Xbf7y/0o&keytype=ref&siteid=fa

hours and vibrated cells at 24 hours appeared to be sebj reorganized. However, spaceflown cells remained growth The identity of the cDNA spots incorporated into the arrested, while vibrated cells resumed active growth microarray GeneFiltersTM (Research Genetics) may be (Lewis et al., 1998). Thus, lymphocyte growth arrest in found at http://www.resgen.com. microgravity is not a direct result of microtubule disruption caused by vibration during shuttle launch. Effect of simulated shuttle launch vibration on However, the reorganized microtubules may not function cytoskeletal gene expression in T lymphocytes (Jurkat) normally. Not all changes in the cytoskeleton may be Approximately 4,300 genes were interrogated by cDNA detectible by fluorescence microscopy yet such changes microarray in Jurkat cells sampled 4, 24, and 48 hours may affect transport of critical signaling molecules or after vibration. (Lewis et al., 2001). Only two

impact other cytoskeletal processes. cytoskeleton-related messages, and C-NAP1, were up-regulated compared to controls in both flown and vibrated cells as shown in Tables 5 and 6. Most differences in gene expression in vibrated compared to Gravitational and Space Biology Bulletin 17(2) June 2004 5 M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview non-vibrated cells occurred four hours after vibration et al., 1998, and Sciola et al., 1999) reported cabling or (Table 6) rather than at 48 hours as in the spaceflown increased numbers of bundled filaments in flown cells. cells. As previously noted, spaceflown cells did not The collapse of the cytoskeleton and increase in plectin proliferate whereas, vibrated cells resumed active growth. gene expression implies plectin's role in restoring light polypeptide (MYL5) was the only cytoskeletal integrity after vibration damage and suggests cytoskeleton-related gene regulated differently at 24 hours possible involvement in reduced cell growth and function in flown cells. All other cytoskeletal genes regulated in space. differently in flown compared to ground controls were Plectin interconnects intermediate filaments, found after 48 hours in microgravity. Reasons for the microtubules, and actin to each other and to the cell delay in expression in microgravity may be related to membrane (Wiche, 1998 and Svitkina et al., 1996) as inadequate transcription or feedback or may reflect shown in Figure 2. It regulates actin filament dynamics inadequate remodeling of the cytoskeleton disorganized (Andra et al., 1998) and functions in cytoskeleton- by launch vibration. membrane anchoring (Smith et al., 1996). Plectin is ubiquitous and provides mechanical strength and structure to cells.

Figure 2. Diagram of plectin interconnecting cytoskeletal elements. Plectin maintains cytoskeletal integrity by linking intermediate filaments, microtubules and actin to each other and to the cell membrane. Plectin also regulates actin filament dynamics and functions as a cytoskeletal-membrane anchoring protein.

Since plectin appears to be involved in reorganizing CONCLUSIONS the cytoskeletion after vibration, the increased expression Possible effects of cytoskeletal anomalies on growth of plectin message at 48 hours in flown Jurkat cells and function of spaceflown cells suggests that even though the cytoskeleton may Cytoskeletal elements in all of the spaceflown cell types reorganize in microgravity, it may not function properly. examined show significant abnormalities. Vassy et al. Whether the work of Papaseit and Tabony demonstrating (2001) reported reduced proliferation of human breast a gravity requirement for microtubule self-assembly in cancer cells (MCF-7) on the Foton orbiting capsule. vitro will hold true for intact cells in microgravity Growth of MC3T3 mouse osteoblasts (Hughes-Fulford remains to be determined. and Lewis, 1996) was retarded and Jurkat cells were growth arrested (Lewis et al., 1998). These data provide The cytoskeleton's role in signal transduction convincing evidence that cytoskeletal anomalies in Normally, microtubules and the cell membrane interact to spaceflown cells may affect proliferation. An intact maintain uniform cell surface tension and cell shape cytoskeleton is essential for cell-cell adhesion and dependent on cytoskeletal polymerization dynamics signaling. (Osborne and Weber, 1976). Hydrostatic pressure Message for the cytoskeletal filament cross-linking changes in low-g can affect membrane-cytoskeleton protein, plectin, was up-regulated in spaceflown and association (Todd, 1989), which in turn, may cause loss of ground-based, simulated launch vibrated Jurkat cells cell surface receptors (Kaplan and Keough, 1982), loss of (Lewis et al., 2001) and in spaceflown kidney cells cellular integrity (Ingber, 1997), and finally disruption of (Hammond et al., 1999, 2001). This strongly suggests that receptor-mediated signal transduction. plectin is involved in recovery of the cytoskeleton from Cells convert physical and mechanical stimuli, vibration damage. Other investigators (Hoeger and including gravity change, into biochemical messengers Gruener, 1990, Gruener et al., 1994, Hughes-Fulford and such as cytokines and . These in turn, trigger Lewis, 1996, Cogoli-Greuter et al., 1997a,b, 1998, Lewis molecular changes including cytoskeletal rearrangement, 6 Gravitational and Space Biology Bulletin 17(2) June 2004

M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview PKC translocation and mobilization of signal transduction microgravity would have serious consequences to the cell molecules, which mediate physiological responses like through improper positioning and signal intracellular transport, growth and differentiation or transduction. apoptosis. Microtubules, cross-linked contractile Cytoskeletal-membrane interactions during signal microfilaments and intermediate filaments, together form transduction require several types of binding proteins. a tension-dependent cellular infrastructure. Messenger One of these, , anchors F-actin to the cell RNA, signaling molecules and other cellular components membrane by linking actin and vinculin (Masuda et al., like enzyme systems and organelles are arranged spatially 1996), transduces signals from membrane receptors to along this scaffolding. The stability of the system contractile proteins, binds actin, , tubulin depends on anchoring of cytoskeletal elements to sites in (Fujii et al., 1997) and a human protein the nucleus and to transmembrane receptors on the cell (MLC-2) (Szymanski and Goyal, 1999), and may also membrane (Ingber, 1997). Interruption of this regulate actin-myosin interactions. is a mechanical structure by launch vibration, or perhaps by peripheral that binds to the - faulty microtubule organization in microgravity, could based membrane cytoskeleton and directly links structural interrupt signal transduction and alter gene expression proteins in the cytoplasm to membrane proteins (Bennett, (Stein et al., 1999) because of decreased mechanical 1982). Several of the genes that regulate cytoskeletal- signaling by the cytoskeletal elements and the cytokeratin membrane association were expressed differently in network (Wang and Stamenovic, 2000). Jurkat cells during spaceflight. These include plectin, Protein kinase C (PKC) is associated with plectin, calponin and an EST similar to ankyrin. Message for all intermediate filaments and stress fibers (Murti et al., of these proteins was increased at 48 hours thus raising 1992). A number of reports link PKC to reduced the question of whether the integrity of the membrane and lymphocyte growth response during spaceflight (Limouse transduction of signals from membrane receptors is et al., 1991, Schmitt et al., 1996, and Hatton et al., 1999). affected in microgravity. Thus, compromise of plectin-cytosketal structural integrity (as evidenced by cabling of cytoskeletal filaments) and the increased expression of plectin Filament polymerization message in microgravity, provide evidence that plectin- Several investigators found shortened cytoskeletal related signal transduction may be affected. Since PKC- filaments in spaceflown cells. This is reported as mediated signal transduction involves trafficking of depolymerized or very short polymers of microtubule signaling molecules along cytoskeletal elements and protein (Piepmeier et al., 1997), discontinuous "points" of signal transduction molecules are positioned and vimentin (Cogoli-Greuter et al., 1997 and Sciola et al., immobilized on the insoluble cytoskeletal scaffold, 1999), shortened microtubules in spaceflown cells (Lewis aberrant plectin-related cytoskeletal organization in et al., 1998), and free tubulin subunits or shortened microgravity could hinder PKC signal transduction. microtubules (Vassy et al., 2001). Other cytoskeleton-associated proteins involved in Messages for several proteins that regulate the signal transduction include and gelsolin, which elongation and polymerization of actin filaments were link cytoskeletal dynamics and signal transduction (Lind expressed differently in spaceflown Jurkat cells (Lewis et et al., 1987). These two proteins regulate actin al., 2001). This raises the question of the possibility of polymerization and filament formation and are modulated faulty filament polymerization in microgravity. The by inositol di-phosphate (PIP) hydrolysis at the cell inner protein gelsolin binds to actin and is a focal point of actin membrane. PIP binds gelsolin (Lind et al., 1987, and Yin polymerization (Kwiatkowski et al., 1985). Plectin et al., 1988), profilin and profilactin (Lassing and functions in actin filament dynamics (Wiche, 1998), and Lindberg, 1985) and interacts specifically with profilin to binds tropomyosin and regulates dissociate the profilin-actin complex and thus mediate interactions between tropomyosin and actin (Fowler, actin polymerization. We found the message for gelsolin 1990). These and reports of decreased filament linearity precursor down-regulated compared to ground controls in (Gruener et al., 1994), reduced number of stress fibers Jurkat cells flown on STS-95 (Lewis et al., 2001) (Hughes-Fulford and Lewis, 1996), microtubules not suggesting the potential for cytoskeleton-related extending to the cell membrane (Lewis et al., 1998), short interruption of signal transduction during an early stage at microtubules and less abundant actin stress fibers (Vassy the cell inner membrane. et al., 2001) all support the possibility that filament Messages for two important organelle positioning and polymerization is affected in microgravity. transport proteins, myosin and , were up- regulated in spaceflown Jurkat cells at 48 hours compared Commonality of gene expression in spaceflown and to ground controls but were not up-regulated in vibrated vibrated cells cells at 24 or 48 hours (Lewis et al., 2001). In both vibrated and flown cells, the cytoskeleton was were, in fact, down-regulated at 4 hours after vibration. disorganized and message for plectin and C-NAP1 were Myosin and dynactin transport molecules and organelles up-regulated (Lewis et al., 2001). This implies a role for to specific locations in the cell and dynactin along with these two genes in reorganizing the cytoskeleton and , a microtubule motor protein, is involved in MTOCs disrupted by vibration. Expression of message positioning the Golgi complex (Burkhardt, 1998). Thus, for the myosin regulatory protein (MYL5) was increased altered motor protein gene expression or transcription in by eleven times in flown compared to ground control yet Gravitational and Space Biology Bulletin 17(2) June 2004 7 M. Lewis – The Cytoskeleton in Spaceflown Cells: An Overview there was no difference in expression of MYL5 between progression of cell cycle phases, signal transduction, vibrated and non-vibrated cells. This strongly suggests tensegrity, metabolism, growth, and gene expression. that some characteristic of spaceflight other that launch We now know that gravity is necessary for the in vitro vibration affected this gene. Whether microgravity per self-orgainzation of microtubules and that there are se, space radiation, electromagnetic fields, or other factors differences in microtubule assembly in low-g and 2g may effect myosin gene expression is yet to be phases of parabolic flight. Research has demonstrated determined. failure of microtubule polymerization and lack of A number of other cytoskeleton-related genes microtubule reorganization in cells in microgravity. including , , dynein, vinculin, , Whether cytoskeletal self-organization in spaceflown spectrin, profilin and were neither up- nor down- cells is impaired remains to be proven. Cells subjected to regulated compared to controls in cDNA microarray simulated launch vibration reform the cytoskeleton and assays of flown or vibrated cells (Lewis et al., 2001). continue to grow, however; spaceflown Jurkat cells This does not necessarily mean that the genes were not appear to reorganize the cytoskeleton but are growth expressed. Since the microarray assay software only arrested even though they are cancer cells. Thus vibration shows values for two-fold increase or decrease in message alone does not account for growth arrest in microgravity. of test versus control samples, these genes may have been Growth arrest must be attributable to some other factor or expressed but not by more than twice the values of cytoskeleton-related abnormality in the microgravity controls. Hughes-Fulford et al. (1996) using RT-PCR, environment. found no significant difference in expression of actin Based on gene expression, two proteins appear to be message in flown MC3T3 mouse osteoblasts. involved in re-organization of the cytoskeleton after vibration damage. Message for plectin, an ubiquitous and Comparison of cytoskeletal genes expressed in multi-functional cytoskeletal linker protein, and the suspension culture cells (Jurkat) and anchorage centriole-associated protein C-NAP 1, were up-regulated dependent human kidney cells. after vibration in ground-based studies and in spaceflown Two landmark experiments using cDNA microarray to cells. Up-regulation of plectin message in flown cells at evaluate the expression of thousands of genes (10,000 48 hours may imply that the cytoskeleton is not properly genes in anchorage-dependent kidney cells, Hammond et organized or that plectin message transcription is faulty. al., 2000; and more than 20,000 genes in the T cell Genes regulated differently in microgravity compared to suspension culture line, Jurkat, Lewis et al., 2001, 2002) ground controls and vibrated cells, identify the most provide a huge database of information that may be likely genes or pathways sensitive to altered gravity and compared to genes expressed by other cell types and for can serve to identify candidate genes and pathways for determining which genes, pathways and processes are future investigations. likely to be affected by spaceflight. Although the type of cDNA microarrays used and sampling times in ACKNOWLEDGEMENTS microgravity (six days for kidney cells and 24 and 48 hours for Jurkat cells) were different, plectin message was The author expresses sincere appreciation to Dr. Luis A. found to be up-regulated in both kidney and Jurkat cells. Cubano for dedicated work in gene analysis and data Again, this implies that plectin is involved in damage compilation. I would like to thank Drs. B. de Sales repair after vibration or that the cytoskeleton may not be Lawless and Edward Piepmeier, Jr. for productive re-organized properly in microgravity. Both cell types collaborations over the years. I express appreciation to also expressed messages for and myosin light Phillip Bowman, and Baiteng Zhao, Hong-Khanh Dinh, chain proteins differently in flown compared to ground and Johnathan Pabalan for cDNA microarray analyses. controls suggesting that some of the same types of genes And finally, I acknowledge the crews of STS-76 and STS- may be involved in gravity response in cells of totally 95, especially Senator John Glenn and Scott Parazinski different origin. Messages up-regulated in Jurkat but not for conducting the 100% successful experiments on STS- in kidney cells include an EST similar to ankyrin, C- 95. This work was supported in part by NASA Grants NAP1, calponin, tropomodulin, and dynactin. Other NAG2-985 and NAGW-812. cytoskeleton-related genes were expressed in kidney but not in Jurkat cells which implies differential responses REFERENCES depending on cell type. Andra, K., Nikolic, B., Stocher, M., Drenckhahn, D., SUMMARY Wiche, G. 1998. 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