MICRO-GRAVITY AND MECHANOMICS Jack J.W.A. van Loon Dutch Experiment Support Center (DESC), Dept.

Oral Cell Biology, ACTA-Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
ABSTRACT
Sensing gravity by non-specialized cells is still puzzling. We dont know where or by which mechanism such cells sense gravity. These questions in gravisensing are not much different from questions in general mechanobiology. Numerous studies have been reported in this field in the last couple of decades. What are the mechanical properties of a cell? Are there differences in mechanical properties between cell types and if so why? How are forces perceived and transduced to a meaningful biological event. Novel techniques such as optical and magnetic tweezers, atomic force microscopy, magnetophoresis and computer modeling make the field of mechano- sensing or perhaps physicomics accessible. A similar approach should also be applied for gravity-related research. This paper addresses the current techniques used in mechanosensing and exemplifies how a cell could sense the relatively weak force of gravity. KEYWORDS: gravity, microgravity, weightlessness, conformational change, space flight, cell mechanics, mechanomics, physicomics, mechanobiology.

Gravitational biology overlaps with or perhaps is part of mechanobiology. In mechanobiology we try to understand the impact of forces on whole systems, single cells, subcellular structures or even individual molecules. In gravitational biology studies we should distinguish experiments that study direct and indirect gravity effects on cells which describes the impact of gravity on cells in response to changes in the external environment like the lack of convection under true weightlessness; see Klaus (2001) in a previous issue of this bulletin or Thevenet et al. (1996) and Brown et al. (2002). The focus of this paper is on direct effects of gravity onto cells. In mechano- or gravitational biological experiments we apply or remove a mechanical force to change something in the cellular machinery to learn about underlying, general, biological mechanisms. The force evokes changes in structural, mechanical and functional properties via changes in e.g. cytoskeletal rearrangements, protein phosphorylation, second messengers, or gene expression. These changes lead to either damage or adaptations of the cell, which can be divided into several steps. First we have mechanoperception to detect a change in the mechanical forces acting upon a cell, presumably through sensors. The sensors signal is relayed, or mechanically transduced, to a cellular response that elicits a response and or leads to mechanoadaptation (see Figure 1). My hypothesis is that all reported changes in ion concentration, protein levels and phosphorylation, changes in potentials, signaling molecules levels, or any biochemical changes are provoked by a mechanical modification somewhere within the cell or on its membrane. There are no biochemical modifications without prior mechanical change. Probably the first descriptions on the role of gravity, i.e. weight, on living systems were given by the Italian Galileo Galilei (1564-1642). He observed the dimensions of the bones from animals of distinct weights and described that the length-to-width ratio is remarkably different between light and heavy animals (Galilei Galileo, 1974). Heavier animals, like elephants have relatively wider bones as compared to for example an antelope. We know now that cellular processes are responsible for this macroscopic difference or adaptation. Somehow the cells within the bone detect its mechanical usage, including weight, and adapts its structure accordingly. Similar processes occur in other systems like plants. One of the first studies within the plant kingdom were by Sir Thomas Andrew Knight who used a water wheel to study the effect of gravity on plants (Knight, 1806). He germinated garden bean seeds for this experiment and showed that plants orient themselves
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INTRODUCTION Gravity acts on masses over a wide range from the size of our Milky Way of about 2.41020 m to the human body of about 2.0100 m. But what about the impact of gravity on single cells with the size in the order of 110-5 m. A difference of about 25 orders of magnitudes with our Milky Way? At a (sub) cellular level the force of gravity is insignificant compared to the three other basic forces in nature; the weak nuclear force, the strong nuclear force and the electrostatic force, that govern the force field within and between molecules inside a cell. Non-gravity related phenomena like thermal noise (kT) or chemical energies are orders of magnitude larger than a 1g acceleration (see Albrecht Buehler 1991). The main difference between a gravitational load and phenomena like Brownian motion is that gravity is continuously acting in the same direction. Over the last decades space flight provided the opportunity to perform microgravity experiments, and a good body of knowledge provides compelling evidence that this seemingly weak force directly impacts single cells as described by Mesland et al. 1987; Brillouet et al. 1995; Demets et al. 1996; Moore & Cogoli 1996; Brinckmann et al. 1999; Gaubert et al. 1999; Cogoli 2002; Clment & Slenzka 2006; Hder et al. 2006. However, it is still unclear how this force impacts especially non-professional cells. ____________________ * Correspondence to: Jack J.W.A. van Loon
Dutch Experiment Support Center (DESC) Dept. Oral Cell Biology, ACTA-Vrije Universiteit van der Boechorststraat 7 1081 BT Amsterdam, The Netherlands Email: j.vanloon@vumc.nl : Web: www.desc.med.vu.nl Phone: +31653700944; Fax: +31204448683

J.J.W.A. van Loon - Micro-gravity and Mechanomics

mechanical force (e.g. gravity) mechanoperception

mechanosensor

cell

mechanotransduction

mechanoadaptation

cell response

(bio)mechanical changes*

(bio)chemical changes

(bio)chemical or morphological changes

mechanobiology (mechanomics) physicomics

* Direct effects of gravity : No biochemical effect without a preceding mechanical change !
Figure 1. Terminology and sequence of events in cell biomechanics. In mechanobiology we study effects of mechanical forces within and/or applied to a cell or parts of it, this can be termed as mechanomics. Extending this to the overall physical environment and processes around and within a cell we might even use the term physicomics.

along the resultant gravity vector resulting from Earth gravity and artificial gravity by rotation. Since Knight, more sophisticated instruments have been developed to apply and study various mechanical forces onto living systems down to the level of single cells. Instruments and methods to study (sub-) cellular and molecular mechanics In the more distant past, mechano-biological studies were generally performed on a tissue level like whole muscle groups or single bones. Recently we see a shift towards smaller entities like single cells or even individual molecules. Lately various instruments and techniques have been developed to study cellular mechanics. Although not extensive, we shall address some of the most common technologies used to qualify and quantify cell mechanical properties. Kenneth Cole published one of the first papers on the practical use of the voltage clamp better known as the patch clamp technique (Cole, 1949). In short, a small pipette is filled with suitable solutions. A metal electrode in the solution conducts electrical changes to a voltage clamp amplifier. This way the transport of ion through individual ion channels in the membrane can be monitored. Although this technique is not used to directly measure cell mechanical properties several studies using this technique have shown mechanosensitive ion channels like the one for calcium; see Autret et al. (2005).

Later, similar pipettes were used for a membrane aspiration technique (Figure 2A) developed by Mitchison and Swann in the early nineteen fifties, in which forces are applied on the cell or vacuole membrane to deform them (Mitchison et al., 1954). The negative pressure sucks a single cell, or part of a cell, into a micropipette which diameter ranges generally from 1 to 10 m. From the microscopical observed shape change of the cell conclusions can be drawn regarding mechanical properties of the part of the cell in the pipette (Figure 3). Forces using this technique start from a few pN, depending on the applied suction and diameter of the pipette used. (Shao et al. 2004). One of the earlier studies directly measuring whole single cell mechanical parameters were performed by McConnaughey and Petersen in the early nineteen eighties using a cell poker (McConnaughey et al., 1980). In these cytotensiometric measurements using attached mouse 3T3 fibroblasts they were able to apply a force of 10 N (10-3 dyne). Current developments lower the applied force to few picoNewton (pN). See also Figure 2B) Coated magnetic beads can be firmly attached to a cell surface. Applying a carefully directed magnetic field to such a system forces the beads to rotate and thus apply a mechanical stress onto that part of the cell surface the beads are attached to (Figure 2C). Wang and coworkers

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Figure 2. Cartoons for the various toola and techniques used in mechano-biology. A: Membrane aspiration. B: Cell poking or membrane indentation. See also for a recent example of a cell poking study the paper by Geitmann in this same issue (2007). C: Application of magnetic beads attached to a cell surface. D: Representation of a standard optical tweezer setup. The beads are trapped with two laser beams. A polymer, like DNA, or a complete cell (see Fig. 6) may be attached between to the beads. By shifting the path of the lasers one can move the beads and apply a force to the interconnecting object. E: Representation of a particle tracking set-up where stained particles within a cell can be monitored using a regular light or fluorescent microscope. F: A squeezing plate or probe. The upper plate can move carefully towards an attached cell. G: An AFM set-up. The cell surface is probed with a tip fixed to a constantly vibrating cantilever. Both height and force can be measured. H: Surface stretch / deformation experiment. Studies are most often performed applying a uni-direction or omni-directional stress. I: Simple representation of a gravity force acting upon a monolayer of cells. See for an overview of gravity related ground facilities van Loon et. al. (1999). J: Simple representation of hydrostatic pressure acting upon a cell monolayer. K: Fluid shear stress acting upon a cell monolayer. L: Vibration acceleration in X-Y direction applied onto a cell monolayer.

introduced this technique in 1993 (Wang et al., 1993). The working principle for deriving mechanical properties from a cell from such a set-up is based on a close relation between the beads attached to cell surface receptors and the underlying cytoskeleton of microfilament stress fibers, intermediate filaments and microtubules (Figure 4). Optical tweezers utilize an optical field gradient to trap micron-sized refractive objects such as polystyrene beads (Figure 2D). This trap can be controlled spatially in three dimensions. Single molecules can be attached to beads or to the cell surface, e.g., via integrins. Displacement of the beads (Figure 5) or attached cells (Figure 6) is measured by a tracking laser beam and / or by video microscopy. The relation between the displacement and force (i.e. strain) provides information of the mechanical properties of the material. Sasaki et al. introduced the technique in 1992 and the work of Arthur Ashkin (1997) promoted its application in biological studies. Magnetic beads were earlier applied in a magnetic tweezer set-up using a single

DNA molecule by Smith et al. in 1992 (Smith et al., 1992).

Figure 3. An aspirated beet root vacuole. Scale bar is 20 m. Picture courtesy of P. G. Petrov and C. P. Winlove, University of Exeter, UK. Gravitational and Space Biology 20(2) June 2007

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Figure 4. Model of the cell envelope as a composite soft shell composed of the lipid-protein bilayer and the associated actin cortex (assumed to form a partially cross-linked network of the semiflexible actin filaments). The force probe is coupled to the actin cortex through integrins. The apical shell of the cell may be coupled to the lower (basal) cell envelope adhering to the substrate. The coupling (represented here by springs) can be mediated by the microtubuli and the intermediate filaments of the cytoskeleton. Pictures taken from Feneberg et al. (2004), with kind permission of the publisher.

Figure 7. Positioning of coleoptiles and a ferromagnetic wedge, magnetized by a uniform magnetic field as viewed from the side (left), and top (right). The shaded zone represents the area of the high gradient magnetic field (HGMF), where the ponderomotive forces affecting starch-filled amyloplasts are equal to or greater than the force of gravity. The relative orientation between the long axis of the coleoptile and wedge was either perpendicular (a) or parallel (b). Pictures taken from Kuznetsov & Hasenstein (1997), with permission from Oxford University Press.

Figure 5. Characterization of the Src reporters, Src kinase or phosphatase activity, in a HeLa cell. Laser-tweezer traction on a fibronectin-coated bead at the upper right corner of the cell (shown on the left) caused FRET responses due to conformational change of the YFP tagged Src substrate peptide and the CFP tagged SH2 domain. White arrow in left frame represents force direction. Pictures taken from Wang et al. (2005) with kind permission from Nature.

Magnetic properties may also be applied to study cell mechanical properties. Kuznetsov and Hasenstein (1997, Figure 7 and 8) applied a ferromagnetic wedge to generate a high gradient magnetic field to dislocate amyloplasts in barley coleoptiles and tomato hypocotyls. The area of application of the gradient within the tissue is limited to areas of interest where intracellular magnetophoretic displacement of diamagnetic particles such as amyloplasts transduce the magnetic force similar to the force of gravity. High gradient magnetic fields can be applied at a larger scale, i.e. to whole organisms for example in a sufficiently large bore of a Bitter magnet. Berry and Geim demonstrated the levitation of various biological organisms including a living frog (Berry et al., 1997). The authors postulated that magnetic levitation might be seen as possible microgravity simulations for biological samples. However, a distinction has to be made between compensating weight and other phenomena such as convection, as compared to real microgravity (Poodt et al., 2005). Besides using external forces as in the previously mentioned techniques, we may also use straightforward observation to track intracellular components (Figure 2E). Molenaar et al. used fluorescently-tagged beads coupled to telomeric complexes to study the dynamic behavior of the telomeric DNA in osteosarcoma cells (Molenaar et al. 2003, Figure 9). Tseng et al. (2002) made use of the inherent Brownian motion of microinjected fluorescent particles in their live-cell multiple-particle-tracking microrheology technique (see Figure 10). They showed that the cytoplasm is much more mechanically heterogeneous than relatively simple reconstituted actin filament networks.

Figure 6. Images of a red blood cell (RBC) being stretched from 0 to 193 pN using an optical tweezer set-up. (a) The left column shows images obtained from experiment while (b) the center column and (c) right column show top view and threedimensional view of the half model corresponding to the large deformation simulation of the RBC, respectively. The color contours in the middle column shows distribution of constant maximum principal strains. Picture from Mills et al. (2004), with kind permission Tech Science Press, Forsyth, USA.

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A fairly commonly used device in biomechanics is the atomic force microscope, AFM. With the advent of the AFM (Binnig et al. 1982, 1986) resolution on the nanometer scale has become possible and its ability to manipulate objects is a significant improvement over light microscopy. AFM works by moving a sharp, micro fabricated tip over a surface while simultaneously recording tip deflection (Figure 2G). The deflection time course is then converted into an image of the surface profile. To probe the mechanical properties of a cell surface, one uses the AFM tip to exert precisely controlled forces in selected locations and records the corresponding displacements. In many ways this method is related to cell poking with larger probes, but it has better spatial and force resolution. The obvious limitation of the technique is that manipulation can only occur through the accessible surface of a cell, i.e. one cannot measure elastic moduli inside the cell without an influence of boundary conditions. One can both indent cells or also pull on cells when the tips are attached strongly enough to the cell surface. Figure 12 shows the difference in compliance of cell with and without vinculin. Although AFM is restricted to the cell membrane and only part of the sub-membrane structures can be scanned. However, recent studies show that the cell internal morphology can be visualized using AFM with scanning near-field ultrasound holography (Shekhawat et al., 2005). From technologies focusing on individual cells we now turn to devices initially used in batch cultures. Clever improvements have been made to some of these systems that comply with the trend toward single cell studies. One often-used paradigm is surface stretch (Figure 2H). A monolayer of cells is grown on a flexible membrane that is stretched or otherwise deformed to apply a mechanical stress via the substrate to the cells. Murray and Rushton developed one of the first systems of this kind used in bone biology (Murray et al., 1990). In this system a culture chamber attached with silicone grease was fixed to

Figure 8. Induction of curvature by HGMF in barley coleoptiles. Curvature was initiated by positioning a ferromagnetic wedge close to the coleoptile tip. The seedlings, wedge, and magnets (not shown) were mounted on a 1 rpm clinostat with the coleoptile growing perpendicular to the horizontal axis of rotation. Presumably due to the action of an HGMF on amyloplasts the shoots curved toward and around the wedge as if responding negatively gravitropic. The image on the right show curvature after 5 h of clinorotation. Pictures taken from Kuznetsov & Hasenstein (1997), with permission from Oxford University Press.

Figure 9. Quantitative analysis of movement of telomeres and centromeres in a single nucleus in a human osteosarcoma cell, U2OS. A: Telomeres and centromeres were visualized using cyan fluorescent protein tagged telomere-associated protein, CFP-TRF2 (red / dark gray) and green fluorescent protein tagged centromere protein A, GFP-hCENPA (green / light gray), respectively. Quantitative movement analysis of telomeres and centromeres can be perforemed of trajectories is shown in B. The average mean square displacement (MSD) values of both telomeres and centromeres in one nucleus revealed that the average MSD of centromeres is similar to that of the population of constrained telomeres. Pictures from Molenaar et al. (2003) and used with kind permission from the EMBL J. / Nature Publishing Group.

A relatively new technology to study cell mechanical compliance or stiffness is a micro-squeezing plate (Figure 2F). With this device it is possible to apply static or dynamic loads up to about 500 N with a resolution of 10N on individual cells, measuring the resulting forces while observing the cell with a confocal microscope. (Peeters et al. 2003). The device is specifically developed to study the cellular background mechanisms for pressure sores (Figure 11).

Figure 10. Multiple particle tracks of microspheres injected into live cells. The thermally excited motion of the particles was video tracked for 20 s. (A) Superposition of a phase-contrast micrograph with a fluorescence micrograph used for the identification and tracking of the probe microspheres. The scale bar is 20 m. (B) Trajectories of microspheres a and b. Pictures from Tseng et al. (2002) and used with kind permission from the Biophysical Society. Gravitational and Space Biology 20(2) June 2007

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biology has long been, and maybe still be, the actual space flight, microgravity or better microweight experiments. If we want to study the impact of weight onto systems, it is as important to go into orbital space flight, as it is relevant to use centrifuges. Although physical processes, and therefore biological systems, are behaving differently under (near) weightlessness (Albrecht-Buehler 1991, Todd 1989) studies under hypergravity c.q. hyper-weight conditions can provide information for the entire gravity spectrum. (See Hatton et al., 2003; Hemmersbach et al., 1999; Searby et al. 2005; Tabony et al., 2004, and van Loon et al., 2007a). Although it was at first argued from a purely physical point of view, that non-specialized single cells might not be susceptible to small force such as gravity (Pollard, 1965), many studies have shown otherwise. In an experiment using an AFM within a large-radius centrifuge we were able to monitor shape changes of the same cell going from 1 to 3g (Figure 15). Not only hypergravity centrifuges but also microgravity simulators such as a clinostat or a random positioning machine are very useful tools in this respect (Briegleb 1965, 1967, 1992; Galland et al., 2004; Klaus 2001; van Loon et al. 2007b). Another paradigm to study cell mechanics properties is by the application of hydrostatic pressure (Figure 2J). This has also been used quite frequently in studies on cell mechanics in the plant and animal kingdom in cell turgor (Geitmann 2007; this issue) or bone / cartilage research, respectively (e.g. Veldhuijzen et al., 1979; Klein Nulend et al., 1987; Figure 16). In a more recent study by Kim and coworkers (2007) hydrostatic pressure was used to study signal transduction pathways and mechanotransducers in mesenchymal stem cells. They showed that cyclic hydrostatic pressure enhances osteogenesis in a 3D culture system via ERK1/2 activation.

Figure 11. Orthogonal projections of a myoblast after 0 and 10 m axial displacement the glass probe. Picture from Peeters et al. (2003), with kind permission from the IEEE Journal Medical and Biological Engineering and Computing.

a gold-coated polycarbonate membrane. The chambers were stretched in one direction by means of a lead screw / lever system attached to a computer-controlled actuator to study the dose (i.e., force)-response relationship of PGE2 release in confluent cultures of primary mouse calvariae cells. Gilchrist and colleagues (2007) studied individual cells using a micrometer device providing a uniaxial stretch to a cell-seeded substrate (Figure 13A). The fluorescent staining of mitochondria and nuclei was applied to monitor cytosol and nuclear strains, respectively. These data showed clearly that the cytosol is more compliant than the nucleus (Figure 13B). Our research community does not need an explanation why we would like to use gravity as one of the environmental parameters to study basic processes in life sciences (Figure 2I). However, the focus in gravitational

Figure 12. Results from AFM measurements in histogram and 3D image from wild type (F9) (A) and vinculin-deficient (5.51) mouse embryonic carcinoma cells (B). As indicated by the grey-scale (on the right), the wild type cell is less deformable by the cantilever than is the 5.51 cell. The lack of vinculin in the 5.51 cell is reflected by a shift toward the softer region (histogram). Picture from Goldmann et al. (1998), with kind permission from the Federation of European Biochemical Societies.

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Figure 13. Schematic of micrometer device accommodated on a light microscope used to apply uniaxial stretch to cell-seeded membranes. Picture from Gilchrist et al. (2006), with kind permission from the Elsevier Publishers.

Although hydrostatic pressure is presumed to act directly onto the whole cell, a recent paper by Charras et al. (2005) showed that there are isolated, local effects at the cellular level. The current general view is that the cytoplasm acts as an incompressible viscoelastic continuum or viscous liquid and that the hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Although the pressure was generated from within the cell, their data suggests that the pressure is not equally distributed throughout the cell. There is a nonequilibration of pressure on scales of a cell i.e. at ~10m within a time period of about 10sec (Figure 17). Besides hydrostatic pressure, osmotic pressure also provides insight in cell mechanics and functioning. Chondrocytes exposed to a hypo-osmotic stress reveal a decrease in elastic moduli and apparent viscosity but changes were not apparent when cells were exposed to hyper-osmotic environment. Rapid changes in cortical actin were noted with hypo-osmotic stress. (Guilak et al., 2002, Figure 18). The study of fluid shear stress to cells has emerged from studies on the vascular system (Figure 2K). While the focus in earlier reports was on the interaction of white blood cells to a vessel wall (Schmid-Schoenbein et al. 1975), recent studies apply fluid shear to unravel signal transduction pathways in endothelial cells (Sun et al. 2007). Fluid shear stress does not only pertain to cardiovascular cells. It has been postulated that fluid shear might also regulate bone cell mechanics (Piekarski et al., 1977; Cowin et al., 1991). Recent studies from our lab have shown that MC-3T3-E1 osteoblastic bone cells are particularly responsive to specific fluid shear stress rates (Bacabac et al., 2004, Figure 19).

Figure 14. Measured longitudinal (Exx) substrate and cell strains in primary cells from the anulus brosus of a porcine intervertebral disc. For each cell, measured cytoplasmic (C, green box) and nuclear (N, red box) strain measurements are shown. Picture from Gilchrist et al. (2006), with kind permission from the Elsevier publishers.

Figure 15. AFM images of part of the same single cell at 1-g (A), 2-g (B) in total scan area of 1116 m. In these AFM cell topography pictures we see part of the cell in the left and upper part of the picture. The dark brown area in A, upper right part marked with arrowhead , is the glass surface to which the cell is attached. The measurements were out of range in the black area in A, the lower right hand corner at . The glass surface is used as reference plane and all cell heights are related to this value. The higher parts of the cell, about 1.2 m, are in the brightest domains. Time between each measurement was 4 min. and 16 sec. Scanning one frame took half of this time. Calibration bar is in m units. Picture reproduced from van Loon et al. (2007), with kind permission of Review of Scientific Instruments. Gravitational and Space Biology 20(2) June 2007

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frequencies between 5 and 100 Hz have shown that nitric oxide and prostaglandin E2 production respond at specific frequencies (Figure 20). These effects might be due to nuclear motion within the cell during vibration (Bacabac et al. 2006a). Naturally occurring vibrations due to Brownian motions can be used to estimate cell compliance as has been shown by Bacabac and colleagues (2006b). Fitzgerald et al. (1996) showed that random vibrations during a Shuttle launch have a significant impact on gene expression in osteoblastic cells.
Figure 16. A: Tissue volume of calcified diaphysis and noncalcified epiphyses per rudiment after 5 days of culture without (control, C) or with the application of Intermittend Compressive Force (ICF). B : 45Ca incorporation into the acidsoluble mineral phase per metatarsal rudiment, after 1 and 5 days of culture in the absence (control, C) or presence of ICF (n = 16). It can be concluded from these experiments that hydrostatic pressure has an anabolic effect on embryonic bone mineralization. Picture from Klein Nulend et al. (1987), with kind permission from the publisher.

In addition to the auditory and cochlear system vibrations play an important role in nature. Vibration at large frequency ranges are used by a variety of sensory systems, like the ear or the proprioceptive sensors in feet or jaws in order to locate pray or for communication (Hill, 2001). The function of these wide range mechanoreceptor systems is to transfer mechanical signals into relevant biological functions. However, vibrations may also be exogenously applied to generate a biological effect. Tanaka et al. (2003) applied Gaussian quasi-white noise to stimulate bone formation. Experiments in our lab using

Also various configurations of cell culture micro patterned substrates are applied in relation to mechanobiology (Balaban et al., 2001). Lemmon and Romer (2007; this issue of the ASGSB bulletin) started to use flexible rod shaped structures fixed onto a culture surface to which cells could attached. The rods would deflect in response to internal outward-directed forces. The level of deflection provides a good estimate of the forces involved. Loesberg et al. (2006, 2007) used a more approach of an isotropic microstructured surface in combination with hypergravity stress or simulated microgravity in a random positioning machine, RPM, to reveal the forces involved in cell orientation and morphology (Figure 21). Properties of standard plastic culture flasks are often taken for granted. Experiments are performed with regular tissue culture substrates, which is quite distant from the actual in situ mechanical environment that cells experience in vivo. Therefore cells are likely to incorporate such external conditions into their physiological response (Georges et al. 2004).

Figure 17. Generation of local blebs in a melanoma cell line. A. Local perfusion of Wheat Germ Agglutinin (WGA) Alexa 488. WGAAlexa 488 was only incorporated in the region of the cell that was exposed to flow from the micropipette. B. Local perfusion of a 300mM sucrose solution. Vacuoles form in the vicinity of the perfused region. C. Local inhibition of myosin II ATP-ase by local perfusion of blebbistatin D. Local inhibition of ROCKI by 3-(4-pyridyl)indole. Solid black lines delineate the flow out of the micropipette. On each image, the inset text gives timing relative to local application of treatment. The normalised blebbing indices show the evolution over time of blebbing in the region exposed to inhibitor and in the free region. Error bars show the standard deviation. Asterisks denote significant changes in the blebbing index when compared to the initial blebbing index. Scale bars=10m. Picture from Charras et al. (2005) with kind permission from Nature.

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Figure 18. SEM of the effects of osmotic stress on chondrocyte morphology. (A) In hypoosmotic medium (153 mOsm), chondrocytes swelled significantly, exhibiting a relatively smooth plasma membrane. (B) In isoosmotic medium (303 mOsm), chondrocytes exhibited numerous membrane ruffles and microvilli. (C) Hyperosmotic medium (466 mOsm) decreased cell volume with an apparent increase in membrane ruffling. Scale bar = 10 m. Picture from Guilak et al. (2002) with kind permission from the Biophysical Society.

Figure 21. Rat dermal fibroblasts grown on a micro-grooved surface. Cells orient differently under simulated microgravity (B) conditions in the random positioning machine conditions in a 24h culture as compared to the 1xg control (A). Picture taken from Loesberg et al. (2006) with kind permission of the publisher; Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

Finally, data emerging from biomechanical experiments of the sort mentioned above can be used to build a virtual cell. One can construct so-called finite element models, FEM, of a single cell or its components. New studies using these models can reveal interesting new insights into mechanical processes that later can be tested in vivo (Prendergast in this issue, 2007). DISCUSSION The last decade has seen a very fruitful contribution by physicists and engineers to biological questions such as in mechano-biology. In line with the terminology for DNA, RNA or protein research, genomics, transcriptomics and proteomics, respectively (see for example Figures 22A+B), we might use the term mechanomics to describe all the mechanical phenomena involved in cell physiology. This term was introduced by Sem et al. (2001). They used nuclear magnetic resonance, NMR technologies that characterize proteinligand interactions combined with bioinformatics strategies across large families of proteins such as kinases and dehydrogenases. The term enzyme mechanomics describes this newly enabled gene family wide characterization of structure function correlations but the term may be applied more widely. Mechanomics could include all mechanical interactions on the level of the cell, tissue and whole organism. On a cellular level, the cytoskeleton with its stress fibers, intermediate filaments and microtubules are likely candidates for force generation and transduction processes. More intricate and possibly localized processes include the mechanical properties between the main cytoskeletal components and connections between the cytoskeleton and the cell membrane or cell wall. Mechanomics would also include molecular motors that use cytoskeletal structures such as myosins, dyneins, prestins or kinesins to facilitate intracellular transport and contractions. Processes like particle invagination, cell division, motility, cytoplasmatic streaming or particular ion channels are largely depending on mechanical or other physical processes. Therefore we should expand the term mechanomics to include other physical parameters such as pressure, temperature, electro-magnetic fields et cetera
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Figure 19. Nitric oxide (NO) production by bone cells (MC3T3) is linearly proportional to the rate of pulsatile uid shear stress (PFSS). The steepest slope was found at 5 min (0.11 PaHz-1), indicating that the highest bone cell response to uid shear stress rate occurs rapidly. Picture from Bacabac et al. (2004) with kind permission from Elsevier.

Figure 20. Effect of vibration stress on PGE2 production by bone cells. Mouse calvarial bone cells, MC-3T3-E1, respond in negative correlation to the applied maximum acceleration rate (max. acc. rate) of vibration stresses from 5, 30, 60 and 100 Hz. Picture taken from Bacabac et al. (2006a) with kind permission of the FASAB J. publisher.

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Figure 22A: An example of a proteomics approach in which protein-protein interaction maps for Saccharomyces cerevisiae to quantitatively address the role of the cytoskeleton in intracellular signaling. This figure demonstrates a network of signaling proteins linked to the cytoskeleton, suggesting a distinct role of the cytoskeleton in signal transduction. The largest connected cluster of 1458 interacting proteins of the database by Uetz et al. (Uetz et al., 2000). In this cluster, yellow (light gray) and green (dark gray) dots denote cytoskeletal and signaling proteins, respectively, as defined by our criteria. Proteins in red (black) are shared by the two subclasses. The analogous cluster in the database of interacting proteins, DIP, network (Xenarios et al. 2000) contains 4198 proteins. It is not shown here because the density of proteins was too high for visual examination.

Figure 23. An example of the 3D structure of an inner membrane bound protein, cdc42. Shown here is a Cdc42 collybistin II complex. Such molecules, as well as transmembranal molecules, might be involved in mechano (gravi-) sensing by changing their conformation in response to mechanical loads. Picture generated from the crystal structure database based on the paper by Xiang et al. (2006) with kind permission from the publisher.

and use a more comprehensive term of physicomics that covers all physical properties involved in cell, tissue or body physiology. Characteristics and interactions of physical properties within a cell should be published similarly as in proteomics studies such as by Forgacs et al. (2004) on signaling networks and the cytoskeleton (Figure 22). While the very first space biology experiments were quite basic and from the look-and-see type, experimental facilities for gravitational biological studies have been improved over the years. However, current facilities and modules for space flight in vitro cell biology studies are still limited to simple rinse-and-fix type studies. Cells are cultured for some time, maybe some media changes are performed and finally stopped with some chemical fixation. The nature of these experiments limits the assays mostly to mechano-transduction and mechano-adaptation but is less capable for studies on mechano-perception as shown in Figure 1. Studying mechanosensing or gravisensing, especially in non-professional cells, depends on flight facilities and the concepts and technology of the devices described here. We need to invest in more sophisticated facilities capable of real time measurements such as confocal and near scanning field microscopes, atomic force microscopes and optical tweezers. Such research facilities would provide a universally usable basis for cell biological research on the International Space Station, as initially promised. However, none of these capabilities have been realized. It seems that the hypothesis based on the various physical properties posed by Pollard (1965) or Albrecht-Buehler (1991) that the force of gravity is too small to provoke direct physiological responses effect on a single, nonprofessional cell is still valid. However, there are other examples in nature where the forces for mechanical loads onto a system, as measured or predicted in vivo, are too small to generate a biological response. It has been shown

Figure 22B: Combined cytoskeleton-signaling subnetwork, derived from the largest connected cluster in A: Yellow (open) and green (gray) dots denote signaling and cytoskeletal proteins, respectively, proteins in red (black) are shared by the two subclasses. Only proteins with at least one connection are shown. Results are only shown for the database by Uetz et al. (2000). Picture taken from Forgacs et al. (2004) with kind permission of the Company of Biologists.

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that bone cells do respond to fluid shear stress in vitro (Piekarski et al. 1977) and that these shear forces may account for bone mechanoadaptation in vivo (Burger et al. 1998). However, the in vitro forces applied are much larger compared to predicted in vivo conditions. In an attempt to bridge this gap for understanding bone mechanoadaptation Han et al. (2004) postulated a 3D model to amplify the mechanical stress by about one order of magnitude. In the auditory system a model is proposed that amplifies the signal for high frequencies by hair bundles in the cochlea (Kennedy et al. 2005), while Brokaw postulated structural models that amplify the strain generated by motor enzymes (1997). Even relativistic models have been proposed to explain the way biological systems might amplify the impact of weak electromagnetic field onto cells (Saxena et al. 2003). So although gravity is a weak force on a cellular level similar amplification mechanisms might play a role in gravisensing, especially in non-professional cells. The nature and localization of the mechano- or gravity sensor are key questions. Although this paper does not focus on the possible molecular candidates for this sensor (see review by Hughes-Fulford, 2004), as hypothesized, this molecule(s) should be able to go through some kind of conformational change. I it is likely to be found in the load-bearing regions of or near the focal adhesion complex and corresponding cytoskeletal connections as shown for cdc42 molecule (Figure 23, Xiang et al. 2006), vinculin (Balaban et al. 2001), fibronectin (Baneyx et al. 2001), angiostatin (Grandi et al. 2006) or PECAM-1 (Osawa et al. 2002). However, other regions within the cell should not be excluded nor the possibility that due to (micro-)gravity mobile parts within the fluidic membrane might be rearranged in such a way that different reaction kinetics might become apparent. It was Tyler in the early days of discovery of the cytoskeleton who postulated that rigid and elastic elements within the cell might be involved in gravisensing (Tyler, 1966). See also Figures 24 and 25. The current assumption that many effects of (micro-)gravity on suspension cultures stem from the lack of convection in near weightlessness (see e.g. Thevenet et al., 1996, Brown et al., 2002 and Klaus 2001), this phenomenon could also apply to attached cells. Gravity is a body force, so the whole cytosol might somehow be involved and this stress might be guided or concentrated to limited areas such as focal adhesions. It has been postulated that the small effect of gravity is integrated and amplified through mechanisms like reaction-diffusion (Turing 1952; Papaseit et al., 2000), stochastic resonance (Pierson et al., 1995; Galvanovskis et al., 1997; Greenwood et al., 2000; Hnggi, 2002 (see also Figure 26) or signal averaging of a constant small stimulus (Kondepudi, 1991). Experiments applying mechanical forces like fluid shear stress or tethered beads exhibit a threshold value of ~ 1nN per cell (Huang et al., 2004). The force to generate a conformational change in individual molecules like FATGravitational and Space Biology 20(2) June 2007

Figure 24. A diagram of a linked system of rigid and elastic elements in which the stability depends on the force exerted by a gravitational receptor (w) and which is independent of direction. Y, rigid rod to which structural framework (not shown) is attached: e. elastic element; II, universal joint: p, pulley-system or frictionless ring: f, flexible, relatively inelastic fiber. Picture from Tyler (1966) with kind permission from Elsevier Publishers.

Figure 25. Confocal microscopy visualization of !-tubulin in a human breast cancer cell line, MCF-7. c: 1g in-flight control, cell fixation 48 h after launch. f t2 g (g, cell fixation 48 h after launch). In the 1g samples we see long, strongly labeled microtubles radiating throughout the cytoplasm, while in the g samples only a few filaments could be distinguished against the strong (gray) background. This more or less diffuse labeling could correspond to either labeled free tubulin subunits or numerous but very short microtubles. Scale bar is 10 m. Picture from Vassy et al. (2001), used with kind permission from FASEB J. publisher.

Figure 26. The principle of thresholds and stochastic resonance: a) Neuronal-like dynamics detect those events that rise above some threshold value (the thin top line). A weak, periodic sub-threshold signal (thick line) can therefore be detected only if its dynamics are assisted by noise (noisy trace). A crossing event occurs most likely when the weak signal assumes its peak value. b) Upward-directed crossing events trigger a firing of spike-train dynamics, u(t). Picture taken from Hnggi (2002) with kind permission of dr. Hnggi and the publisher.

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Paxillin is 125pN (Kamm et al., 2004). The maximum force that can be generated due to the weight of an attached model cell, i.e. half sphere with diameter of 10 m and a density 1.1103 kg/m3, is about 5 pN. Simple Newtonian mechanics imply that 1 nN or 125 pN is equivalent to about 200 and 25 times the force resulting from unit gravity, respectively. The effect of gravity on non-professional cells suggests that no straightforward linear relation exists between the magnitude of this force and its impact on cells. ACKNOWLEDGEMENTS I want to thank Dr. Millie Hughes-Fulford and Dr. Karl Hasenstein for their valuable suggestions and critical review of this paper. This paper was possible with support from NWO-ALW via the Netherlands Institute for Space Research, SRON grant MG-057. REFERENCES Albrecht-Buehler, G. 1991. Possible mechanisms of indirect sensing by cells. ASGSB Bulletin 4(2): 25-34 Ashkin, A. 1997.Optical trapping and manipulation of neutral particles using lasers. PNAS USA 94: 4853-4860 Autret, L., Mechaly, I, Scamps, F., Valmier, J., Lory, P., Desmadryl, G. 2005. The involvement of Cav3.2/!1H Ttype calcium channels in excitability of mouse embryonic primary vestibular neurones. J Physiol 567(Pt 1): 67-78 Bacabac, R.G., Smit, T.H., Mullender, M.G., Dijcks, S.J., Van Loon, J.J., Klein-Nulend J. 2004. Nitric oxide production by bone cells is fluid shear stress rate dependent. Biochem Biophys Res Commun 315: 823-9 Bacabac, R.G., Smit, T.H., Van Loon, J.J., Doulabi, B.Z., Helder, M., Klein-Nulend, J. 2006a. Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm? FASEB J 20(7): 858-64 Bacabac, R.G. 2006b. Microrheology of mechanosensitive bone cells. In Academic thesis Bone Cell Mechanosensitivity and Microgravity. Free University Amsterdam, The Netherlands, January 24 Balaban, N.Q., Schwarz, U.S., Riveline, D., Goichberg, P., Tzur, G., Sabanay, I., Mahalu, D., Safran, S., Bershadsky, A., Addadi, L., Geiger, B. 2001. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 3(5): 466-72 Baneyx, G., Baugh, L., Vogel, V. 2001. Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer. PNAS USA 98(25): 14464-8 Berry, M.V., Geim, A.K. 1997. Of flying frogs and levitrons. Eur. J. Phys 18: 307313 Binnig, G., Rohrer, H., Gerber, Ch., Weibel, E. 1982. Surface studies by scanning tunneling microscopy. Phys Rev Lett 49: 57-61 Binnig, G., Quate, C.F., Gerber, C. 1986. Atomic force microscopy. Phys Rev Lett 56: 930-33 Briegleb, W. 1965 Ein Beitrag zur Frage physiologischer Schwerelosichkeit. In: Deutsche Luft- und Raumfahrt Vortrge aus dem Institut fuer Flugmedizin gehalten auf dem VI Internationalen und XII Europaeischen Kongress fuer Luft- und Raumfahrtmedizin in Rom 1963 und dem XIII Internationalen Kongress fuer Luft- und Raumfahrtmedizin in Bublin 1964 (Briegleb W. Eds.) Cologne, DVL 37-42 Briegleb, W. 1967. Ein Modell fuer SchwerlosichkeitsSimulation an Mikroorganismen. Naturwissenschaften 54(7): 167 Briegleb, W. 1992. Some qualitative and quantitative aspects of the fast-rotating clinostat as a research tool. ASGSB Bulletin 5: 23-30 Brillouet, C. 1995. Biorack on Spacelab IML-1. Mattok, C., Ed. SP-1162. ESA Publication Div. ESTEC, Noordwijk, The Netherlands Brinckmann, E., Brillouet, C. 1999. Biorack on Spacehab. Perry M., Ed. SP-1222. ESA Publication Div. ESTEC, Noordwijk, The Netherlands Brokaw, C.J. 1997. Mechanical components of motor enzyme function. Biophys J 73(2): 938-51 Brown, R. B., Klaus, D., Todd P. 2002 Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli. Microgravity Sci Technol 13(4): 24-9 Burger, E.H., Klein-Nulend, J., Cowin, S.C. 1998. Mechanotransduction in bone, molecular and cellular biology of bone, in: M. Zaidi, E.E. Bittar, O.A. Adebanjo, C.L.H. Huan (Eds.), Advances in Organ Biology, JAI Press, Stamford, Connecticut, USA: 123-136 Charras, G,T., Yarrow, J.C., Horton, M.A., Mahadevan, L., Mitchison, T,J. 2005. Non-equilibration of hydrostatic pressure in blebbing cells. Nature 435(7040): 365-369 Clment, G., Slenzka, K. (Eds.) 2006. Fundamentals of Space Biology: Research on Cells, Animals, and Plants in Space. Springer, New York, USA Cogoli, A. Edt. 2002. Cell Biology and Biotechnology in Space. Adv. Space Biology and Medicine. Elsevier Cole, K.S. 1949. Dynamic electrical characteristics of the squid axon membrane. Arch. Sci. Physiol 3: 253-258

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