1. Introduction
Earth’s gravitational force has been a constant condition during Earth’s history and the evolution of cells and organisms [
1]. The human organism reacts and partially adapts to altered gravity [
2,
3] in a time frame of a few hours up to several weeks [
4]. The cellular response to microgravity, such as functional alterations [
5,
6], nucleus size, and shape [
7,
8,
9], and RNA transcription [
10,
11] mostly occurs within seconds, sometimes months [
12,
13,
14,
15]. The question about the mechanism by which gravitational force is transmitted into a biological process was raised decades ago [
16,
17], but it was rarely addressed in experimental approaches. For the “sensing” of gravity in eukaryotes [
18], cytoskeletal processes and mechanosensitive ion channels have been discussed, but the presented theories remained speculative and were not substantiated by systematic experiments. Therefore, none of the hypotheses of gravitational force sensing and transduction into a biological process in mammalian cells has been confirmed by experimental data so far.
Gravitational force (
FG) is one of the four fundamental physical forces in nature, has an attracting character, and correlates linearly with an object’s mass. Newton’s law of gravity describes
FG as the product of two objects’ masses (
m1,
m2) attracting each other, divided by the square of the distance (
r), and scaled by the gravitational constant (
G):
FG =
G(
m1m2/
r2). To calculate the gravitational force exerted on an object on Earth, this formula can be simplified by setting the distance between the center of the Earth and the object equal to the Earth’s radius (
R⊕) and by combining
G, Earth’s mass (M
⊕), and
R⊕ into the gravitational acceleration constant
g = 9.81
m/
s2, yielding
FG =
mg. As described by Einstein in his work on general relativity, there is no difference between the observation of a mass falling down in a steady reference system (related to as gravitational mass) and a resting mass in a reference system that is linearly accelerated towards the mass (related to as inertia mass). As consequence, the gravitational force exerted on an object is only measurable if the reference system is not accelerated in the same way, as for instance the International Space Station (ISS) in low Earth orbit. The ISS’s actual gravitational acceleration,
g, that would be measurable if it was not accelerated, is only reduced by 8% due to the slightly greater distance to the Earth’s center [
17], yet objects inside experience weightlessness relative to the station. As a sum of many effects, every platform that supplies weightlessness has a residual gravitational force, usually between 10
−3 and 10
−6 g [
19,
20]. The residual gravitational force can be due to vibrations caused by on-board machinery, atmospheric drag, solar radiation pressure, and gravitational attraction between the test object and the platform [
21]. Depending on the mechanism that is studied, different quality levels of microgravity are required. If a platform supplies microgravity at a value below the mechanism’s threshold, it is also called functional weightlessness [
22].
In weightlessness, hydrostatic pressure (as a result of weight of the water column on top of a reference point) and sedimentation of particles in a solution (as a result of gravitational force and buoyancy minus Stokes’ friction) are absent. Entropy-driven diffusion of particles resulting from temperature dependent Brownian motion is present, whereas concentration dependent (Raleigh) convection as a cause of buoyancy is absent. Therefore, mixing of liquids is affected by altered gravity [
17].
All these changes occur in altered gravity environment and have the potential to affect biological processes. Due to the restriction of microgravity flight platforms, ground based facilities (GBFs) have been developed to simulate microgravity conditions in biological experiments [
20].
Since all GBFs are ground based, none of them is able to provide true force-free conditions. By averaging forces, those devices attempt to simulate microgravity. Due to the logistical and cost advantages, the popularity of GBFs is high, although the validity of GBFs in terms of producing results comparable to flight-induced microgravity platforms remains uncertain. Clinostats rely on the principle of constantly turning a sample, thereby averaging out the gravitational force vector around one (1D/2D clinostat) or two (3D clinostat) axes. Technically speaking, the gravitational force still acts on the sample, but the direction of the gravitational vector on the sample is changing constantly. This is why the terminology "vector-averaged gravity" (vag) is usually preferred over "simulated microgravity” [
23]. Whether the achieved results of a system compare to its behavior in microgravity depends on the particular effect of interest [
24]. The first 2D clinostats were used to study plant gravitropism [
25], in which a rotation speed of several seconds per revolution is sufficient to simulate force-free conditions [
26]. In general, rotation induces centrifugal forces that act like additional gravitational forces and depend on the radius. Thus, fast rotating clinostats for cell culture experiments have a very small diameter that minimizes the distance from the center to the sample, usually in the range of millimeters [
20]. Nevertheless, in addition to the centrifugal forces, Coriolis force is also acting [
22]. Further, 3D clinostats are a more recent development whereby the system can independently rotate around a second axis, therefore allowing access to all three dimensions. This allows averaging forces in all directions, potentially further decreasing effects of gravitational sensing [
27]. Random positioning machines (RPMs) are 3D clinostats that randomly change rotation speed and direction, which is supposed to further randomize the gravity vector possibly beneficial for averaging out gravitational effects [
28,
29]. However, the random movement changes seem to induce shear stress into samples [
30]. Rotating wall vessels (RWVs) are devices with liquids in horizontal rotation where the speed is tuned to match the sedimentation speed of the contained objects, for example cells [
31]. As consequence, cells experience a constant mode of falling downwards (sedimentation) with shear forces induced by the friction between the liquid and the objects [
32]. Since sedimentation is limited by this friction, cells still experience a (reduced) gravitational force. Because an averaging of orientation may not be achieved for particles with anisotropic mass distribution, RWVs are only suitable for certain types of applications, but not for achieving vag in cellular liquid cultures. The stable levitation of diamagnetic samples like aqueous samples, and therefore most biological materials, allows true microgravity simulation, since the gravitational force is compensated for by generating a counteracting force in every point of an object and not only on the surface like for example in an RWV, preventing any internal force [
33]. However, the required strong magnetic fields of several Tesla are likely to heavily influence cellular processes [
34]. The wide use of clinostat microgravity simulators is based on the assumption that most biological processes have longer integration times than the period of gravitational vector rotation (e.g., 1 s for 60 rpm 2D clinorotation). The faster the studied mechanism acts, the higher the rotation frequency must be set to guarantee conditions in which the gravitational force vector is evenly distributed in all directions. Since the discovery of ultrafast reactions to altered gravity, there is debate on the effectiveness of this approach [
35].
Several mechanisms induced in microgravity are also sensitive to hypergravity [
3,
4]. Fixed-angle centrifuges are well established and affordable platforms to generate hypergravity and 1×
g control conditions in microgravity experiments [
36]. The underlying physical principle is that objects on a circular trajectory experience centrifugal (pseudo-)force (because the rotating frame of reference is no physical inertial frame). This force (
FZ f) is dependent on the rotation angular frequency (ω = 2π
f), the radius (
r) from the center, and the object’s mass (
m):
FZ f =
mω
2r =
m 2π
f r. With known dimensions of the centrifuge the (simulated) gravitational force can be tuned by varying the rotation frequency. Centrifuge setups with adherent cells are prone to shear forces, if the geometry of the vessel where cells are rotated in does not match the rotation circle (for example for flat bottom vessels) [
17,
37]. Additionally, during centrifugation, Coriolis force occurs: If an object moves linearly in an axis perpendicular to the rotating axis, the reference system (test tube/dish) moves a bit further. From the rotating reference system’s point of view, moving objects therefore exhibit a curved trajectory. The force is defined
with
m being the object’s mass,
ω = 2
π f being the centrifuge’s rotation angular frequency and
v being the object’s speed. In a liquid, molecules and particles move around freely which is described by Brownian motion and therefore being influenced by the Coriolis force. As a consequence, particles diffuse much slower during ultracentrifugation and show an elliptic and no more ball-shaped diffusion pattern [
38]. This effect is smaller by many orders of magnitude for slow rotation, e.g., when simulating slightly elevated gravitation levels, but could play a role for biological macromolecules, during cellular signaling. Therefore, centrifuges are not an ideal simulation of hypergravity. However, compared to ground based “simulators” for microgravity, this platform is much closer to the real conditions.
Although several types of cultured cells are sensitive to gravity [
39,
40], the immune system belongs to the most affected systems during spaceflight (reviewed in [
41,
42,
43]). Sensitivity of human immune cells to reduced gravity has been confirmed in numerous studies in flight-induced and simulated microgravity (reviewed in [
11,
43]). Immune system weakening during long-term space flights could contribute to an increased susceptibility to infections, autoimmunity, and cancer during exploration class missions. Thus, it is indispensable to understand the cellular and molecular mechanisms by which altered gravity changes genomic stability and gene regulation in cells of the immune system and to assess mechanisms for adaptation. Changes of the gravitational environment induce strong alterations of human physiological systems, which respond and adapt within hours and weeks [
44]. At the cellular level, changes of the gravitational force affect morphology, proliferation, differentiation, signal transduction, and gene expression [
45] and have been detected within seconds in isolated cells of the immune system [
10,
12,
13,
46,
47]. We recently investigated the dynamics of gene expression response to different gravitational environments in human Jurkat T lymphocytes in combination of parabolic flights with a suborbital ballistic rocket experiment and with control experiments for excluding all possible other factors of influence [
13,
14]. In these experiments, we detected a significantly high number of differentially regulated transcripts after 20 s of microgravity exposure [
13], and gene clusters which are stable in different gravity environments [
14]. According a recent comparison, the biological responses of cells in suspension in a 2D clinostat is similar to those in flight-induced microgravity [
20]. We therefore aimed to validate this current state of the art opinion by systematically comparing transcriptomics data from human Jurkat T cells in microgravity provided by a suborbital ballistic rocket mission with vag provided by a 2D clinostat and hypergravity provided by 9×
g centrifuge experiments. Thus, we performed ground based studies in a 2D clinostat and a hypergravity centrifuge using comparable experimental conditions to the parabolic flight and suborbital ballistic rocket experiments. Our aim was also to investigate if the underlying mechanisms for the perception and transduction of the gravitational force into chromatin is faster or slower than the time needed in a 2D clinostat to average gravitational force in all directions once, meaning one revolution per second. If the transcriptome effect of flight-induced microgravity can be reproduced in a 2D clinostat, the process of primary gravity perception must take longer than 1 s.
3. Discussion
We analyzed the influence of altered gravity on the gene expression response of non-activated Jurkat T lymphocytes using the GBFs fast rotating 2D clinostat and a 9×
g centrifuge. Both platforms hold the same type of pipettes containing cell samples. During 5 min of vag in the clinostat, we found 768 significantly upregulated and 373 significantly downregulated TCs compared to 1×
g controls, while 5 min of 9×
g hypergravity induced 3046 significantly upregulated and 2732 significantly downregulated TCs. The baseline control (1×
g control vs. BL) revealed 2125 significantly upregulated and 496 significantly downregulated TCs, reflecting the influence of filling of the cell suspension into the pipettes and releasing the cell suspension from the pipettes (
Table 2). Eliminating those significantly differentially expressed TCs due to pipette usage yielded 644 upregulated and 227 downregulated TCs after 5 min of vag, and 2753 upregulated and 1843 downregulated TCs after 5 min of 9×
g hypergravity (
Table 3). The significantly differential gene expression response ranged between −4.00 and +4.28-fold changes.
We also analyzed the gene expression in non-activated human Jurkat T lymphocytes in microgravity and hypergravity during the TEXUS-51 sounding rocket campaign. The suborbital ballistic flight provided 75 s of hypergravity during the rocket launch followed by 5 min of flight-induced microgravity. Exposure of Jurkat cells to 5 min of hypergravity in the 9×
g GBF centrifuge and to hypergravity of the rocket flight resulted in 42 differentially regulated TCs respectively, 28 annotated TCs being significantly differentially expressed using both research platforms (
Figure 8 and
Figure 12,
Table 6). A functional gene ontology analysis of the associated genes revealed an association of the genes to transport, cytosol, nucleotide binding, Poly(a) RNA binding, nuclear speck, RNA binding, intracellular membrane-bounded organelles, and regulation of alternative mRNA splicing via the spliceosome (
Table 7).
When we compared the vag -sensitive TCs of the GBF experiment with the flight-induced microgravity-sensitive TCs of the suborbital rocket flight, 11 TCs respectively (five annotated TCs) were significantly altered using both research platforms (
Figure 9 and
Figure 13). Among these annotated five TCs are: (1) the G3BP1 (G3BP stress granule assembly factor 1) enzyme, that unwinds DNA and RNA duplexes in an ATP-dependent manner, (2) KPNB1 (karyopherin subunit beta 1) which is involved in nucleocytoplasmic transport of e.g. ribosomal proteins or H1, H2A, H2B, H3, and H4 histones, (3) NUDT3 (nudix hydrolase 3) involved in nucleoside phosphate metabolic pathways and negatively regulating the ERK1/2 pathway, (4) POMK (protein-O-mannose kinase), which is involved in forming transmembrane linkages between the extracellular matrix and the exoskeleton, and (5) SFT2D2 which seems to participate in the fusion of retrogradely transported endosomes with the Golgi complex (
Table 6 and
Table 9). A functional gene ontology analysis of these TCs showed no significant association to cellular functions or processes.
The analysis of hypergravity and microgravity double-sensitive TCs in both platforms revealed four annotated TCs associated with 4 genes: (1)
G3BP1, (2)
KPNB1, (3)
NUDT3, (4)
SFT2D2, all of which have already been identified as vag/flight-induced microgravity-sensitive (
Table 6).
Interestingly, the direct comparison of hypergravity and vag/flight-induced microgravity-sensitive TCs between the platforms showed that the GBF experiments revealed about five times as many hypergravity as vag-sensitive TCs. Furthermore, the 2D clinostat and 9×
g centrifuge experiments showed more up than downregulated TCs. We could not observe this effect in the data of the TEXUS-51 mission. Here, no major differences between the number of hypergravity and flight-induced microgravity-sensitive TCs were observed, and the differentially expressed TCs were rather downregulated instead of upregulated (
Figure 5).
Our results indicate that less than 1% of all examined transcripts show the same response in vag and flight-induced microgravity. Even when only considering the 396 transcripts differentially expressed in flight-induced microgravity, less than 12% show the same response in flight-induced microgravity and vag. Based on these surprising results, we have reinvestigated the forces prevailing in a 2D clinostat, commonly accepted to be classified as simulated microgravity. To simplify the calculations for our system, we assumed that human Jurkat T cells in suspension behave like small particles.
The study of particle behaviors in flows is a fundamental problem in fluid dynamics and a number of theoretical, computational, and experimental studies have investigated the trajectory of spherical particles in purely rotational flows. Given the dimensions of the cells and clinostat, only studies pertaining to low Reynolds number flows are relevant in this context. Lin and colleagues [
48] theoretically analyzed the trajectories of particles in rotational flow at small but finite Reynolds numbers and demonstrated that buoyant particles converge to a stable equilibrium position located under the rotational plane in accordance with experimental observations [
49]. In contrast, particles denser than the surrounding fluid do not have a stable equilibrium point and ultimately follow a divergent trajectory, spiraling outward. Brought back to the dimensions of the problem at hand, the spiraling motion of the particle over 5 min would not markedly deviate from the circular motion of the surrounding media.
In shear and rotational flows, fluid inertia further causes the particle to spin. For small Taylor numbers (
) (with r: radius, ω: angular velocity, µ: viscosity, p: density) the angular velocity of the particle (
) relates to that of the fluid as follows:
In our application, Ta is in the order of . The particle angular velocity can thus be considered equal to that of the clinostat.
Summarizing the above,
Figure 14 illustrates the general motion of a spherical particle matching the characteristic dimension and density of human Jurkat T cells over one revolution of clinorotation. As particle and clinostat have the same rotation rate, the gravitational force and associated hydrostatic pressure gradients follow a cyclic pattern in the particle reference frame, for the gravitational force with a null average, and for the pressure with an average of 15 Pa over one revolution per second. In contrast, the centrifugal force and resultant hydrostatic pressure gradients, as well as shear stresses associated with the outward radial displacement of the particle, have a constant direction in the particle reference frame (
Figure 14d). However, as stated earlier, it should be kept in mind that the maximal centrifugal force in the clinostat is 0.006×
g and therefore very small compared to the normal unidirectional gravitational force.
In contrast to real microgravity environments, cells in a clinostat still experience gravitational forces albeit with a constantly changing direction (
Figure 14c,d). The Jurkat T cells in our experiment are constantly exposed to oscillating forces with maximum changes of 29.4 Pa, 2.3 mPa, and 17.5 × 10
−12 N for pressure, shear stress, and gravitational force, respectively (
Figure 14). For a rotation speed of 60 rpm, the direction of gravitational forces integrated over 1 s (corresponding to one revolution of the clinostat) are averaged. According to the clinostat theory [
20,
30], the biological effect of gravitational forces is canceled, if the initial trigger mechanism requires at least one second of stimulation, corresponding to one full 360-degree rotation in a 2D clinostat rotating with 60 rpm. Because the transcriptome response during the microgravity phase provided by a suborbital ballistic rocket flight was not detectable in vag provided by a 2D clinostat, it is unlikely that the initial trigger of gravitational force transduction into the chromatin lasts longer than one second.
Our experiments are limited by the inherent limitation of ballistic trajectories, the launch and hypergravity phase, which is preceding always the microgravity phase. Although we applied four types of control experiments (
Figure 1), not controlled artifacts induced by the preceding force conditions cannot be fully excluded. In our experiments, only less than 1% of the transcriptome changes in flight-induced microgravity could be reproduced by clinorotation. Our data suggest that the initial trigger and perception mechanisms of gene expression changes in microgravity is faster than one second. Indeed, mechanical force transduction into the chromatin occurs within milliseconds [
50], allowing the nuclear structure to respond directly without biochemical signaling [
51,
52]. In previous studies which have investigated the oxidative burst reaction in mammalian macrophage cells in microgravity, rapid adaption to a microgravity environment was detected in microgravity on board of the ISS [
10], but not in the vag of clinorotation experiments [
12,
53,
54].
If 99% of such pivotal events cannot be simulated by analogous clinostat experiments, the general assumption that 2D clinostats provide a valid simulation of microgravity and can be recommended for most biological organisms [
20] should be revised. If in addition the primary trigger of gravity-induced gene expression response [
10,
46] is less than one second, the fundamental question of suitability of the clinostat system for the investigation of mammalian cells arises. We therefore recommend that sufficient validation experiments should be carried out before starting experiments with vag as simulation for real microgravity.
Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces occurring during cell culture experiments in general.