Space Flight Affects Motility and Cytoskeletal Structuresin Human Monocyte Cell Line J-111

Maria Antonia Meloni,1 Grazia Galleri,1 Giuseppe Pani,1,2 Angela Saba,1 Proto Pippia,1

and Marianne Cogoli-Greuter3*1Department of Physiological, Biochemical and Cellular Science, University of Sassari, Sassari, Italy2Radiobiology Unit, Molecular and Cellular Biology expert group, Belgian Nuclear Research Centre, SCK�CEN, Mol, Belgium3Zero-g LifeTec Zurich, Switzerland

Received 21 July 2010; Revised 5 December 2010; Accepted 8 December 2010Monitoring Editor: George Bloom

Certain functions of immune cells in returning astro-nauts are known to be altered. A dramatic depressionof the mitogenic in vitro activation of human lympho-cytes was observed in low gravity. T-cell activationrequires the interaction of different type of immunecells as T-lymphocytes and monocytes. Cell motilitybased on a continuous rearrangement of the cytos-keletal network within the cell is essential for cell–cellcontacts. In this investigation on the InternationalSpace Station we studied the influence of low gravityon different cytoskeletal structures in adherent mono-cytes and their ability to migrate. J-111 monocyteswere incubated on a colloid gold substrate attached toa cover slide. Migrating cells removed the colloid gold,leaving a track recording cell motility. A severe reduc-tion of the motility of J-111 cells was found in lowgravity compared to 1g in-flight and ground controls.Cell shape appeared more contracted, whereas the con-trol cells showed the typical morphology of migratingmonocytes, i.e., elongated and with pseudopodia. Aqualitative and quantitative analysis of the structures ofF-actin, b-tubulin and vinculin revealed that exposureof J-111 cells to low gravity affected the distribution ofthe different filaments and significantly reduced the flu-orescence intensity of F-actin fibers. Cell motility relieson an intact structure of different cytoskeletal elements.The highly reduced motility of monocytes in low grav-ity must be attributed to the observed severe disruptionof the cytoskeletal structures and may be one of the rea-sons for the dramatic depression of the in vitro activa-tion of human lymphocytes. VC 2011 Wiley-Liss, Inc.

KeyWords: immune cells, locomotion, microgravity, Inter-national Space Station

Introduction

Twenty-five years of research in space and in modeledlow gravity on ground, provided either by a fast rotat-

ing two-dimensional clinostat or a random positioningmachine (RPM), have clearly shown that mammalian cellsare showing alterations in their structure and function af-ter exposure to altered gravity conditions [Cogoli andCogoli-Greuter, 1997; Lewis, 2002]. Cells of the immunesystem are the most severely affected. Investigations on theeffect of gravity on peripheral blood lymphocytes havebeen triggered by reports in the early 70s that the respon-siveness of lymphocytes from cosmonauts and astronautstowards mitogens was remarkably reduced after spaceflight[Konstantinova et al., 1973; Kimzey et al., 1975; Kimzey,1977].A first space experiment with isolated human lympho-

cytes in culture revealed that their proliferative response tothe mitogen Concanavalin A (Con A) was suppressed bymore than 90% compared to the ground control (GC)[Cogoli et al., 1984]. An intensive follow-on researchclearly showed that exposure of T lymphocytes in cultureto low gravity conditions is accompanied by a major in-hibitory effect, remarkably reducing their mitogenic acti-vation process and severely altering growth rate, signaltransduction, cytokines production, gene expression, cytos-keletal structures and motility [for reviews see: Cogoli,1993, 1997; Cogoli and Cogoli-Greuter, 1997; Lewis,2002; Cogoli-Greuter, 2004; Cogoli and Cogoli-Greuter,2007]. Human lymphocytes have been found to undergoapoptosis by exposure to modeled low gravity [Maccar-rone et al., 2003]. Furthermore it has been clearly demon-strated that factors other than low gravity (i.e.,accelerations and vibrations during launch and cosmicradiation) can be excluded to be responsible for thedepressed activation of lymphocytes.

Maria Antonia Meloni, Grazia Galleri and Giuseppe Pani havemade equal contributions to this work

*Address correspondence to: Marianne Cogoli-Greuter, Zero-gLifeTec GmbH, Riedhofstrasse 273, Zurich 8049, Switzerland.E-mail: marianne.cogoli@zeglit.ch

Published online 13 January 2011 in Wiley Online Library(wileyonlinelibrary.com).

RESEARCH ARTICLECytoskeleton, February 2011;68:125–137 (doi: 10.1002/cm.20499)VC 2011 Wiley-Liss, Inc.

125 n

The mechanism of T cell activation is very complexand still not yet fully understood. Three signals arerequired for full T cell activation. The first signal is deliv-ered to the T cell receptor (TCR)/CD3 complex either bythe antigen presenting cell, or by anti-CD3, or by themitogen Con A. The second signal is a costimulatory sig-nal delivered either by accessory cells – usually monocytes– via B7/CD28 interaction [for a detailed review seeSmith-Garvin et al., 2009] or by anti CD28. Many cellsurface receptors are able to enhance signaling through theTCR/CD3 complex, but CD 28 is the most efficient[Acuto and Michel, 2003]. After stimulation of the TCR/CD3 complex and CD28 the signal is transferred to thenucleus, resulting in the synthesis of interleukin-2 (IL-2)[Leonard and O’ Shea, 1998]. IL-2, acting as third signal,is secreted and bound to its receptor (IL-2R). This indu-ces further synthesis of IL-2 and its receptor, resultingfinally in full activation [reviewed in Bachmann and Oxe-nius, 2007].The activation process consists of several steps in which

specific cytokines are secreted, locomotion of T cells isreduced and the structure of different cytoskeletal ele-ments is altered before the onset of cell division. Conceiv-ably, gravitational forces may interact with cell organellesand structures like the cytoskeleton, having significantdensity differences [Cogoli, 2006].So far it is still not known which structures or mecha-

nisms might act as ‘‘gravity responders’’ in lymphocytes,but there is increasing evidence suggesting that inhibitionof lymphocyte proliferation is due to alterations occurringwithin the first few hours of exposure to low gravity[Hashemi et al., 1999; Hughes-Fulford et al., 2005].Recently it has been discovered that an impaired induc-tion of early genes regulated primarily by transcriptionfactors NF-jB, CREB, ELK, AP-1 and STAT contributeto T cell inhibition in modeled low gravity [Boonyarata-nakornkit et al., 2005]. Furthermore protein kinase A(PKA) signal transduction was found to be down-regu-lated in modeled low gravity, whereas the PI3-K and PKCsignals were not inhibited.Cell–cell interaction and aggregate formation are im-

portant means of cell communication and signal deliveryin the mitogenic in vitro activation of human T lympho-cytes. Especially the interaction between T cells andmonocytes is essential for the delivery of the costimulatorysignal. Lack of the costimulatory signal results in anergy, acondition in which T cells can no more be stimulated. Inearlier flight investigations we observed in real time thatlymphocytes in the presence of Con A were highly motileand formed aggregates [Cogoli-Greuter et al., 1996], butno dates are available so far on the motility of monocytesin low gravity. During locomotion, the cytoskeletal struc-tures within the cells are subjected to repeated cycles ofreassembly processes. The observed changes in activationand signal transduction as well as motility and aggregate

formation of lymphocytes in low gravity may be relatedalso to structural changes in the cytoskeleton due to grav-ity unloading. Marked alterations in the structure of theintermediate filaments of vimentin [Cogoli-Greuter et al.,1998] as well as in the microtubules network [Lewiset al., 1998] were observed in Jurkat cells – a T cell line –after exposure to low gravity.In the present investigation on the International Space

Station we studied the motility of adherent human mono-cytes J-111 and alterations in the cytoskeletal structures ofF-actin, b-tubulin and vinculin in these cells in low grav-ity. As we had found that suspended T lymphocytes arehighly motile in low gravity, we hypothesized that animpaired motility of human monocytes could hinder thedelivery of the costimulatory signal to activate the B7/CD28 pathway, and thus could be one of the reasons ofthe observed loss of T cell activation in low gravity. Thishypothesis is further supported by the findings that a cos-timulation of CD3-activated cells by CD28 antibodies inmodeled low gravity results in a normal T cell activation[Vadrucci et al., 2005].Monocytes play an important role in the adaptive

immune defense, where they act as antigen-presenting cellsand deliver the costimulatory signals essential for a fullactivation of T lymphocytes. Because of their phagocyticactivity monocytes are also fundamental for the innateimmune system. Furthermore monocytes migrate from theblood into other tissues and differentiate into macro-phages and dendritic cells.In preparation for this experiment we have investigated

the motility of monocytes and changes in their cytos-keletal structure, specifically F-actin, b-tubulin and vincu-lin, in modeled low gravity conditions provided by theRPM using a similar protocol as for the flight experiment[Meloni et al., 2006].

Materials and Methods

The space experiment Motion and Interact (MIA) hasbeen performed on the International Space Station in theframe of the Kubik Bio 1 mission of the European SpaceAgency. Kubik is an incubator for space experiments man-ufactured by Comat Aerospace (Toulouse, France). A si-multaneous control experiment was done at 1g in space(Kubik centrifuge). Furthermore a GC experiment wasperformed with the same batch of cells.

Experiment Hardware

The MIA Hardware has been developed and constructedby EMPA (Dubendorf, Switzerland) and Doctor DanyLightweight (Urdorf, Switzerland). Each MIA unit (40 �13.3 � 20 mm3) can hold one glass cover slide (23 � 12mm2) covering a culture chamber (volume: 980 ll) andcontains culture medium and fixative in their respective

n 126 Meloni et al. CYTOSKELETON

special compartments (volume: 270 ll each) [Cogoli-Greuter et al., 2005]. By a special manual mechanism thefixative was brought into contact with the medium in theculture chamber. All parts of the units have been provento be fully biocompatible in tests with J-111 cells.

Cell Line and Cell Culture

J-111 is a monocyte/macrophage cell line derived fromhuman acute monocytic leukemia, obtained from ‘‘Speri-mental Zooprofilatic Institute’’, Brescia (Italy). The cellsdisplay a good adhesion capacity and a certain extent ofepithelial morphological polymorphism related to differentfunctional and metabolic status of the cell. The cells weregrown in RPMI-1640 medium GlutaMAX containing10% fetal calf serum (FCS), 4-(2-Hydroxyethyl) pipera-zine-1-ethanesulfonic acid (HEPES) 20 mM, sodium bicar-bonate 5 mM, gentamycin 50 lg/ml and were subculturedevery 3 days using 0.25% trypsin/ethylenediamine tetraace-tic acid (EDTA) (all obtained from GIBCO, Invitrogen,Carlsbad, CA). The cells for experiment MIA were trans-ported in culture flasks at 37�C from Sassari to Baikonour.

Experiment Sequence Test

In order to test the flight protocol proposed by the spaceagency we have performed an experiment sequence test inmodeled low gravity using the RPM. Two identical set ofsamples (cells in the MIA hardware) were exposed at 25�Cfor 60 h to modeled low gravity, simulating the transfer ofthe cells to the International Space Station, followed by 24h at 37�C either at modeled low g (simulating 0g in space)or 1g (simulating in-flight 1g control; 1gSF). GC sampleswere incubated first for 60 h at 25�C followed by 24 h at37�C. Viability of the cells was evaluated with trypan Blueand the state of nuclei with 4’,6-diamidino-2-phenylindolehydrochloride (DAPI) staining (Table II). All values, viabil-ity and state of nuclei of single cells, are expressed in per-centage on total amount of cells. The counting wasperformed manually under inverted microscope for viabil-ity and on fluorescence images for state of nuclei. For theevaluation of the state of the nuclei we counted only cellswith the correct circularity of nuclei.

Locomotion Assay

A quantitative assay for the motility of spreading mamma-lian cells in culture, which are associated with large surfaceextensions, i.e., lamellipodia and filopodia (microspikes)has been described by Albrecht-Buehler and Lancaster[1976]. In short, freshly suspended cells, plated on top ofa gold particle-coated microscope glass slide, produce vari-ous surface protrusions and remove the particles within aring around each cell [Albrecht-Buehler and Goldmann,1976]. During their spreading the cells begin to movewhile cleaning more particles out of their way. The par-ticles around the cells are mostly cleaned out by surface

protrusions during the first hour after plating and becomepartly internalized (phagocytosed) and partly accumulatedon the cell surface in big clumps without being phagocy-tosed [Albrecht-Buehler and Lancaster, 1976]. To distin-guish locomotion on plain surfaces from this combinationof phagocytosis and cellular displacement we better indi-cate this phenomenon as ‘‘phagokinetics’’ and the particlefree tracks conveniently visualized as ‘‘phagokinetictracks’’. In order to observe and quantify the motilebehavior of monocytes under different gravity conditionswe plated J-111 cells onto microscope glass cover slidescoated with colloidal gold [according to Albrecht-Buehlerand Lancaster, 1976]. Exposure of the cells to gold par-ticles had no obvious toxic effects as proven by prelimi-nary viability tests using trypan blue dye exclusion test(Sigma Aldrich, St. Louis, MO). J-111 cells showed nor-mal growth and spreading on gold coated glass slides.

Gold Coating

Glass cover slides were first incubated with bovine serumalbumin (BSA) (10 mg/ml tridistilled H2O) at room tem-perature for 10 min, then quickly washed with ethanol100% and exposed to 85�C for 10 min. Subsequently 5ml of the gold suspension (AuCl4H 1.45 mM; SigmaAldrich, St. Louis, MO) at 60–80�C were added. After 45min of incubation, the slides were washed in normal saltsolution (113 mM NaCl, 3 mM KCl, 1 mM MgCl2, 15mM Na-phosphate, ph 7.8 in tridistilled H2O), and theninserted into the MIA units. The units were brought toBaikonour at room temperature.

Preflight Activities in Baikonour

For the MIA experiment three identical sets have been pre-pared: one set for exposure to low gravity, the second forthe 1g control in space and the third for the GC. Each setcontained two units with gold-coated glass cover slides forthe evaluation of cell motility. The other four units withuncoated glass cover slides were used for the investigationof the influence of low gravity on different cytoskeletalstructures. The culture chambers of all MIA units werefilled two days before launch with J-111 cells resuspendedin RPMI-1640 with 10% FCS (units with gold coatedslides contained 6.4 � 103 cells, the other 4 � 104), reser-voir 1 with culture medium and reservoir 2 with fixativeparaformaldehyde (22.65%). The units were than incu-bated for 24 h at 37�C to allow the J-111 cells to adhere tothe glass cover slide. The two flight sets were handed over14 h before launch and transferred into the Soyuz rocket.The samples were stored at room temperature duringlaunch and ascent. The GC set was brought to Moscow.

In-Flight Operations

On flight day 3, the samples were transferred into theRussian segment of the International Space Station and

CYTOSKELETON Cell Motility and Cytoskeleton in Microgravity 127 n

loaded into the KUBIK incubator, one set in static posi-tion (low gravity) and one on the centrifuge (in-flight 1gcontrol). After an incubation of 24 h at 37�C all sampleswere fixed with paraformaldehyde (final concentration:4%). The GC experiment was performed in Moscow witha delay of 40 min. The samples were stored at 3–4�C(except for descent and landing) until analysis performedin Sassari. The temperature conditions of the space sam-ples and of the GCs were recorded with the help of aSmartButton data logger (Atal BV, The Netherlands) fromlaunch-11 h (March 29, 2006) until sample return onApril 9, 2006.

Postflight Processing

After landing the samples were delivered at 4�C to Moscowand transported to Sassari together with the samples fromthe GC. The glass cover slides were taken out from eachMIA unit and analysed either for migration tracks or thecytoskeletal structures of F-actin, b-tubulin and vinculin.

Quantitative Analysis of Migration Tracks

To analyze the motility of the J-111 cells, migration trackswere visualized by Bright-field illumination differentialinterference contrast (Normansky) microscope (OlympusOptical, Hamburg, Germany) magnified with a 20� objec-tive. To evaluate the cell displacement image data were col-lected using an F View II Image camera with charge-coupleddevice (CCD) coupled to the software ‘‘ANALYSIS’’ (bothfrom Soft Imaging System GmbH, Munster, Germany).

Statistical Analysis

After grouping displacement ranks the frequency percen-tages (percentage of cells showing a displacement in the dis-tinct group 1–11) and standard deviation have beencalculated. Data were analysed by one-way analysis of var-iance following rank sum test using SIGMA STAT program(Systat software, San Jose, California). The presented dataare the average from 50 cells per slide for each gravity con-dition (low gravity, 1g in-flight and GC), whereby twoslides per condition have been evaluated. Statistical signifi-cance was accepted at the P � 0.0001 level.

Analysis of Cytoskeletal Structures

Cytoskeletal structures of b-tubulin and vinculin weredetected by indirect immunofluorescence techniquewhereas F-actin was revealed by direct fluorescence. Afterextensive washing and cell permeabilization with TritonX-100 (Sigma) 0.1% in phosphate buffered saline (PBS)for 3 min, fluorescent staining was performed by exposingthe slides either to monoclonal anti-b-tubulin clone TUB2.1, (diluted 1:100 in phosphate buffered saline withoutcalcium and magnesium (PBSO), Sigma) or to monoclo-nal Anti-Human Vinculin clone hVIN-1 (diluted 1:100in BSA-PBS, Sigma), both at 37�C for 1 h in a moist

chamber. After washing in PBSO and PBS, respectively, asecond layer of anti-Mouse IgG fluorescein isothiocyanate(FITC) conjugated (diluted 1:100 in BSA-PBSO or BSA-PBS, respectively), was applied for 45 min, at roomtemperature in the dark. For the cytochemical labeling ofF-actin, permeabilized cells were stained with 5 lg/mlPhalloidin-FITC or tetramethylrhodamine isothiocyanate(TRITC) conjugated (Sigma) solution in PBS, at 37�Cfor 15 min. Nuclei were stained with DAPI (BohringerMannheim GmbH, Germany ), (100 ng/ml in PBS) for 6min. Slides were rinsed in PBS and mounted withIMMU-MOUNT (Shandon, Pittsburgh, Pennsylvania,PA). Cytoskeletal structures were visualized by fluores-cence inverted microscope (Olympus Optical, Hamburg,Germany) using an oil immersion 100� objective (NA1.3–0.6). To support the qualitative microscopic observa-tions on cell morphology and cytoskeletal features quanti-tative analysis of cell surface area and fluorescenceintensity of cytoskeleton components of F-actin and b-tubulin were performed on cell images in gray color (eightbit). To obtain not overexposed pictures with good depthof field for fluorescence intensity analysis, the objectiveiris diaphragm was set in order to have N.A. 1 and a sta-tistical analysis about exposure time (data not shown)between all experimental points for b-tubulin and F-actinwere fixed at 4.3 and 3.5 sec, respectively.The morphometric analysis on cytoskeletal structures

was based on algorithm applicable to fixed and immunola-beled cells expressing fluorescently tagged cytoskeletal pro-teins [Lichtenstein et al., 2003]. Fluorescence intensity offilaments of F-actin and b-tubulin were detected by DataAnalysis-algorithm, after background removal and cellperimetral and fluorochrome pixel selection. The dataindicate the rate of labeled actin or tubulin filamentsreferred to the total cell surface and the quantitative extentof cytoskeletal filaments network. Simultaneously, on thesame images, the surface areas of the cells were evaluatedusing a measurement function of the same software. Thepresented data were from at least 50 values per each ex-perimental condition (low gravity, 1g in-flight and GC).Statistical analysis on the quantitative data consisted of aT-test to compare matched values and was supported byMann Whitney test.

Results

For the MIA experiment three identical sets have beeninvestigated. On the International Space Station one setwas placed in the static position in the Kubik incubatorand thus exposed to low gravity in order to evaluate theinfluence of weightlessness on the motility of J-111 cellsand their different cytoskeletal structures. The second setwas placed simultaneously on the Kubik centrifuge for the1g control in space. The 1g control in space is very im-portant in order to distinguish between the effect of low

n 128 Meloni et al. CYTOSKELETON

gravity and possible effects caused by other factors asaccelerations and vibrations due to launch or cosmic radi-ation. The cells exposed to low gravity have the same his-tory related to launch and cosmic radiation as the one at1g in space. A GC experiment with the same batch ofcells (prepared in Baikonour) was performed in the MIAhardware with 40 min delay to the operations in space atthe Institute of Biomedical Problems in Moscow.

Cell Motility

The qualitative microscopic analysis of the migrationtracks of J-111 monocytes on gold particle coated coverslides revealed a normal pattern of cell migration at 1g in-flight and in the GC, similar to those described in the lit-erature [Horwitz and Parson, 1999]. Cells had the typicalmorphology of migrating monocytes, elongated and withpseudopodia and areas around cells appeared completelycleaned out of gold particles (Figs. 1b and 1c, bottom).On the other hand, J-111 cells exposed for 24 h to lowgravity in space showed only very short migration tracks(Fig. 1a, bottom). Thus the motility of monocytes is verymuch reduced under this condition.A quantitative analysis of the migration tracks revealed

a clear difference in the locomotion ability of the cells inlow gravity compared to 1g in-flight and GCs. Cellsexposed for 24 h to low gravity moved with an average of8 lm and the most frequent displacement (43%) wasbetween 0 and 4 lm (Fig. 1a, top), whereas in the in-flight 1g control an average displacement of 31 lm wasobserved and the most frequent displacement (14%) wasbetween 30 and 34 lm (Fig. 1b, top). The cells in theGC showed a displacement of 49 lm on average with themost frequent displacement (40%) of �50 lm (Fig. 1c,top).A remarkable difference of the motility of the cells in

the in-flight 1g and the GC was found. The most likelyexplanation for this discrepancy is that this is due to amemory effect of the cells as they have been exposed for55.5 h to low gravity conditions during ascent and trans-fer into the International Space Station before they wereplaced on the 1g centrifuge. Similar phenomena havebeen observed by us [Pippia et al., 1996] and otherresearch teams [Hatton et al., 2002] in human T lympho-cytes and human monocyte cell line U937 in earlierspaceflight experiments.The results from the MIA experiment on locomotion

of J-111 monocytes exposed for 24 h to low gravity inspace are comparable to those obtained in a previousground based study in the RPM where the cells wereexposed to modeled low gravity [Meloni et al., 2006].Again a normal pattern of monocyte locomotion wasfound in the control samples, whereas very short migra-tion tracks were observed after 24 h of exposure to mod-eled low gravity (Table I).

The quantitative measurement of the surface area of J-111 cells showed a significant difference between lowgravity samples vs. in-flight 1g (P ¼ 0.0112) and lowgravity samples vs. GC (P ¼ 0.0075) revealing that theareas of the cells exposed for 24 h to low gravity condi-tions are significantly smaller than those of the controls(Fig. 2b). In fact, the shape of the cells exposed to lowgravity appeared more contracted, whereas the cells of thein-flight 1g and GCs showed the typical morphology ofmigrating monocytes, i.e., elongated and with pseudopo-dia. Cells exposed to low gravity showed only in somecases very short protrusions.In order to assess cell adhesion and spreading of J-111

monocytes we examined visually samples from the differentgravity conditions (Fig. 2, left panel). No significant differ-ences concerning cell-substratum adhesion were observedbetween cells exposed to low gravity (Figs. 2a-0g) and con-trol samples (Figs. 2a-1gF and 2a-GC), but the cells at 0gappeared smaller and more contracted. This is in agreementwith the quantitative analysis of cell surface area (Fig. 2b).In space no viability tests could be performed before fixationof the cells. Thus, in order to assess the state of the cells, wehave analysed the morphology of the nuclei in the mergedfluorescence images (Fig. 3). In all three experimental condi-tions, DAPI staining showed normal nuclei in about 80%(610% SEM) of the cells. DAPI test performed on eachculture supernatant showed a nearly total absence ofdetached cells. These findings are in agreement with the pre-vious analysis on cell viability in the MIA units in an experi-ment sequence test performed in preparation of the spaceexperiment (Table II).

Cytoskeletal Architecture

A 24 h exposure of J-111 cells to low gravity conditionsin space resulted in severe alterations of the structures of

Table I. Migration Tracks of J-111 MonocytesObserved in Low Gravity in Space and in

Modeled Low Gravity Obtained on the RPMa

and Respective Controls

Gravityconditions

Averagedisplacement

(lm)

Mostfrequent

displacement

Space experiment

0g 8 43% between 0 and 4 lm

1gF 31 14% between 30 and 34 lm

GC 49 40% > 50 lm

Experiment on RPMa

0g 8.7 37% between 0 and 4 lm

GC 59.7 62% > 50 lm

RPM, random positioning machine; 0g, low gravity in space andmodeled low gravity; 1gF, 1g in flight; GC, ground control.aMeloni et al., 2006.

CYTOSKELETON Cell Motility and Cytoskeleton in Microgravity 129 n

F-actin, b-tubulin and vinculin. In 1g conditions (groundand in-flight 1g controls) the structures of all three differ-ent cytoskeletal elements showed a normal and well organ-ized network.

Microfilaments: F-Actin

In the in-flight 1g and GCs the architecture of F-actinappeared normal with a well organized network of cyto-

solic bundles and elongated and extended filopodia (Figs.3a-1gF and 3a-GC). Conversely, a remarkable decrease ofthe density of the filamentous biopolymers of F-actin wasobserved in J-111 cells exposed to low gravity (Fig. 3a-0g). The actin filaments showed a disappearance of thecomplex cytosolic network and appeared mostly localizedclose to the plasma membrane. Identical changes in thestructure of F-actin were observed in cells exposed for 1 hto modeled low gravity on the RPM [Meloni et al.,

Fig. 2. (a) Monolayer of J-111 monocytes from three different gravity conditions: low gravity (a-0g), 1g in-flight (a-1gF) andground control (GC; a-GC). Axiovert 25 Zeiss microscope with phase contrast. (b) Quantitative analysis of J-111 cell surface area(lm2) after 24 h of exposure to low gravity (0g), 1g in-flight (1g) and in GC. Significant differences were observed, by digitalfluorescent microscope, between 0g vs. 1g in-flight (P ¼ 0.0112) and 0g vs. GC (P ¼ 0.0075). All values are expressed as Mean 6SEM.

Fig. 1. a–c bottom. Migration tracks on gold particle-coated glass cover slides of J-111 cells observed with bright-field illuminationdifferential interference contrast (Normansky) microscope after 24 h of exposure to low gravity (a), 1g in-flight (b) and ground con-trol (GC) (c). Very short migration tracks were observed at low gravity and cell shape appeared more contracted (a). Conversely, 1gin-flight (b) and ground samples (c) showed areas around cell completely cleaned out of gold particles and the typical morphology ofmigrating cell, elongated and with pseudopodia. a–c top. Displacement frequencies (in %) of J-111 cells on gold particle-coated glasscover slides after 24 h of exposure to low gravity (a), to 1g in-flight (b) and in GC (c). Cells exposed to low gravity moved between0 and 4 lm with the most frequent displacement (43%) (a) compared to 1g in-flight control (b) with the most frequent displace-ment (14%) between 30 and 34 lm and to the GC (c) with the most frequent displacement (40%) � 50 lm. The data are the aver-age from 50 cells per slide for each gravity condition, whereby two slides per condition have been evaluated. Statistical significancewas accepted at the P � 0.0001 level.

n 130 Meloni et al. CYTOSKELETON

Fig. 3. Immunofluorescence images of F-actin (a), b-tubulin (b) and vinculin (c) in J-111 cells cultured under different gravityconditions. (a) F-actin monochromatic and merge immunofluorescence images with F-actin (green color) and nuclei (blue color) inJ-111 cells after 24 h of exposure to low gravity (a-0g), 1g in-flight (a-1gF) and in ground control (GC; a-GC). (a-I) Fluorescence in-tensity of F-actin filaments at low gravity (0g), 1g in-flight (1gF) and in GC. A statistical significant difference was observed betweenF-actin fluorescence intensity of samples exposed to low gravity conditions compared to 1g in-flight (0g vs. 1gF, P ¼ 0.03) andmostly between 0g samples compared to GCs (0g vs. GC, P < 0.0001). All values are expressed as Mean 6 SEM. (b) b-tubulinmonochromatic and merge immunofluorescence images with b-tubulin (green color) and nuclei (blue color) in J-111 cells after 24 hof exposure to low gravity (b-0g), 1g in-flight (b-1gF) and GC (b-GC). (b-I) Fluorescence intensity of b-tubulin at low gravity (0g),1g in-flight (1gF) and in GC (GC). Statistical difference was not observed between b-tubulin fluorescence intensity of samplesexposed to low gravity conditions (0g) compared to 1g in-flight (1gF), as well as between 1g in-flight compared to GCs, whereas lowgravity samples showed a slight difference versus GCs (P ¼ 0.03). All values are expressed as Mean 6 SEM. (c) Vinculin monochro-matic and merge immunofluorescence images with Vinculin (green color), F-actin (red color) and nuclei (blue color) in J-111 cellsafter 24 h of exposure to low gravity (c-0g), 1g in-flight (c-1gF) and in GC (c-GC).

2006]. After 24 h in modeled low gravity an initial reor-ganization of the actin network was observed.

Microtubules: b-Tubulin

The microtubules of J-111 monocytes showed a normalstructure in the ground as well as in the in-flight 1g con-trol (Figs. 3b-1gF and 3b-GC). They appeared orderlyradiating from the perinuclear area throughout the cyto-plasm toward the cell periphery. Incubation of J-111 cellsin low gravity conditions resulted in a disruption of theb-tubulin architecture (Fig. 3b-0g). The microtubules, re-sponsible for cell division, did not display their typical ra-dial array; they were highly disorganized, and showed amore evident thickening in perinuclear position and a sur-rounding arborization that appeared organized but withshort and incomplete prolongations towards the plasmamembrane. Very similar changes in the structure of micro-tubules were observed in cells which experienced modeledlow gravity for 1 and 24 h in the RPM [Meloni et al.,2006].

Vinculin

Vinculin is an anchor protein which specifically partici-pates in the formation of a submembrane ‘‘plaque’’ struc-ture (focal adhesion plaque) responsible for theattachment of actin filaments to the plasma membrane.Fluorescence images of J-111 monocytes provide profilesof vinculin spots localized in focal adhesion plaques. Inthe in-flight 1g and GCs (Figs. 3c-1gF and 3c-GC) en-dogenous vinculin appeared concentrated as streak-likestructures exhibiting a normal radial orientation towardsthe cell membrane according to cell spreading and con-nected to actin filaments. Samples exposed to low gravity(Fig. 3c-0g) showed vinculin proteins thickened close tothe cell membrane as globular clusters, losing radial orien-

tation and arranging parallel to cell membrane, concomi-tantly to the altered redistribution of actin filaments. Thisis in correlation with the cell morphology showing morecontracted and round cells, as a consequence of thedecrease of cell spreading. Vinculin spots appeared to belarger and the accumulation of fluorescent protein-taggedvinculin in the focal adhesions was usually more promi-nent in control cells (Figs. 3c-1gF and 3c-GC) than incells submitted to low gravity (Fig. 3c-0g). The number offocal adhesions per single cell and the spatial extent ofeach individual adhesion plaque appeared to be reducedin low gravity as compared to those in cells exposed tonormal gravity. The percentage of cells with altered vincu-lin location was 73.8 6 9% in low gravity, and was theaverage from 50 cells per slide, whereby two slides havebeen evaluated. On the contrary samples at in-flight 1gand in the GC showed a normal vinculin distribution (936 7% in-flight 1g and 97.8 6 3% in the GC,respectively).A quantitative analysis revealed that exposure of J-111

cells to low gravity conditions for 24 h affected the cyto-skeleton network not only in the distribution of the fila-ments but also in the fluorescence intensity of thecytoskeletal pattern of F-actin and b-tubulin when com-pared to in-flight 1g and GCs. The morphometric analysisperformed by measuring fluorescence intensity of F-actinor b-tubulin filaments and referred to the total cell surfacearea, showed a significant statistical difference between F-actin fluorescence intensity of cells exposed to low gravityconditions compared to in-flight 1g controls (0g vs. 1g in-flight, P < 0.05) (Fig. 3a-I). The difference was evenmore evident between low gravity samples compared toGCs (0g vs. GC, P < 0.0001). On the other hand, thefluorescence intensity of b-tubulin related to total cell sur-face area showed no statistical difference between b-tubu-lin fluorescence intensity of samples exposed to lowgravity conditions compared to in-flight 1g controls aswell as between in-flight 1g compared to GCs, whereaslow gravity samples showed a slight difference versus GCs(0g vs. GC, P < 0, 05) (Fig. 3b-I).The data of this space experiment and the earlier one

in the RPM [Meloni et al., 2006] suggest that the reducedmigration response in J-111 cells exposed to low gravity(real and modeled) is linked to changes of the cytoskeletalstructures and focal adhesion plaques.

Discussion

The response of any organisms to gravity depends ulti-mately on functions at the cellular level, as single cellfunctions may be affected by gravity themselves. Still littleis known about the effect of gravity at the scale of singlecells, as research in cell biology in space has been rare andfrequently unrepeated. More insight on the influence oflow gravity on single cells comes from investigations

Table II. Cell Viability and State of Nucleiof J-111 Monocytes in Space Investigation

and Mission Sequence Test on RPM

Gravityconditions

Viability trypanblue test (%)

State of nucleiDAPI staining (%)

Space experiment

0g – 76.3

1gF – 81.2

GC – 84.6

Mission sequence test on RPM

0g 78.5 79.2

1gSF 80.5 85.4

GC 85.4 87.8

RPM, random positioning machine; 0g, low gravity in space andmodeled low gravity; 1gF, 1g in flight; 1gSF, 1g simulated flight; GC,ground control.

n 132 Meloni et al. CYTOSKELETON

performed in modeled low gravity on Earth using differ-ent simulation devices. As cells are sensitive to mechanicalforces, low gravity might act on stress-dependent cellchanges and specifically on the cytoskeleton. The cytoskel-eton has been described to be the structure through whichthe cells sense gravity [Ingber, 1999]. The cytoskeletonhas an important role in the maintenance of the cell struc-ture, cell movement and migration. Cell migration is anessential characteristic of many biological processes withinan organism. In previous studies in modeled low gravity,several type of cells, such as T lymphocytes [Sundaresanet al., 2002], human mesenchymal stromal precursor cells[Gershovich and Buravkova, 2007], vascular endotheliumcells [Romanov et al., 2000; Scheglovitova et al., 2000;Vincent et al., 2005] and malignant human MCF-7 cells[Li et al., 2009] showed reduced cell motility and migra-tion. So far the basic mechanisms responsible for thesephenomena in low gravity are still unclear.On Earth, cells have evolved distinct mechanisms for

generating cell movement. One mechanism responsible forcell motility and changes in the shape of a cell is a contin-uous rearrangement of cytoskeletal structures mostly by anassembly and disassembly of microfilaments and microtu-bules. Investigations in low gravity in space and in mod-eled gravity on ground revealed that different cytoskeletalstructures, both in adherent and nonadherent cells, arehighly sensitive to gravity changes [Lewis et al., 1998;Schatten et al., 2001; Vassy et al., 2001; Uva et al., 2002;Cogoli-Greuter, 2004; Li et al., 2009].Monocytes play an important role in the immune

defense. Because of their phagocytic activity and their abil-ity to differentiate into antigen-presenting cells monocytesparticipate in the innate as well as the adaptive immuneresponse. As antigen presenting cell monocytes areinvolved in the activation of T lymphocytes, but they arealso delivering the second signal – a costimulatory signal– via B7/CD28 interaction [Smith-Garvin et al., 2009].Furthermore monocytes migrate from the blood intoother tissues and differentiate into macrophages.Migration of immune cells (lymphocytes and mono-

cytes) is a crucial process during a multitude of physio-logical and pathophysiological conditions such asdevelopment, defense against infections and wound heal-ing [Lauffenburger and Horwitz, 1996; Horwitz andParson, 1999].In the present investigation performed on the Interna-

tional Space Station we found by a quantitative analysisthat in low gravity the locomotion capability of J-111 cellson gold particle coated cover slides is remarkably reducedcompared to the in-flight 1g and GCs. The cell shapeappeared more contracted, whereas the cells of the 1g in-flight and GCs showed the typical morphology of migrat-ing monocytes.The different cytoskeletal structures are very important

for cell motility. Recent findings highlight the cytoskeleton

crosstalk during cell motility, coordination of membraneand actin cytoskeleton dynamics during filopodia protru-sion and the importance of the intact networks of bothcytoskeletal actin filaments and microtubule dynamics incell movements [Horwitz and Parson, 1999; Yang et al.,2009]. Cell migration begins with an initial protrusion orextension of the plasma membrane at the leading edge ofthe cell, driven by the polymerization of actin filaments,and stabilized through the formation of plasma membraneadhesive complexes, regulated by the combined microtu-bules activity. Actin polymerization plays also an impor-tant role in multiple and crucial aspects of the immuneresponse including the antigen recognition, signal trans-duction, T cell proliferation, migration and adhesion[Reicher and Barda-Saad, 2010]. Microtubules are alsoregulators of focal adhesion and turnover of focal com-plex. This is critical for the continued remodeling andreorganization of adhesion contacts during cell migration[Enomoto, 1996]. Microtubule fiber dynamics arerequired for the polarized protrusion of lamellipodia thatdrives the directional cell migration [Hu et al., 2002].The acquisition of cell polarity induced by a reorganiza-tion of the different cytoskeletal elements is very impor-tant for migration, activation and apoptosis in leukocytes,i.e., in lymphocytes and monocytes [Fais and Malori,2003].The cytoskeleton is important for maintaining cell

shape and coordinating cell motility. Furthermore it alsolocates organelles and cellular proteins in their proper spa-tial position with respect to each other and thus also playsa key role in signal transduction [Janmey, 1998]. A dis-ruption of the cytoskeletal network, caused for instancealso by gravity changes, has an impact on signal transduc-tion, cell growth and metabolism [Valitutti et al., 1995;Ullrich et al., 2008]. In the present work we haveobserved severe alterations in the structure of F-actin, b-tubulin and vinculin in J-111 monocytes exposed for 24 hto low gravity conditions compared to in-flight 1g andGCs.The F-actin network showed a remarkable decrease in

the filamentous biopolymers density. The microfilamentsdid not form into their usually strong bundles, with nopreferential orientation and less lamellipodia. Concomi-tantly the normal polyhedral cell shape changed to aroundish one, the long filopodia were no longer present,and thus the cells appeared more contracted with onlyshort protrusions. The layer of actin underneath theplasma membrane, responsible for the cell shape and theproduction of filopodia, was no longer continuous but in-terrupted at various places. The altered shape of the cellscorresponded to the altered network of actin filaments.Similar changes in the actin network, especially a disor-

ganization and reduction of the stress fibers, in cellsexposed to low gravity are also found in osteoblasts[Hughes-Fulford and Lewis, 1996], human vascular

CYTOSKELETON Cell Motility and Cytoskeleton in Microgravity 133 n

endothelial cells [Buravkova and Romanov, 2001], glialcells [Uva et al., 2002], HUVEC cells [Carlsson et al.,2003] and several other cells [for a review see Ullrichet al., 2008]. Microfilaments in MCF-7 cells were notorganized in bundles and the cells lacked lamellipodia [Liet al., 2009].The changes observed in the microtubules network of

J-111 cells in space and in modeled low gravity [Meloniet al., 2006] were similar to those observed by Lewis et al.[1998] in Jurkat cells exposed to low gravity in space,showing that microtubule organizing centers were poorlydefined. The intermediate filament network, responsiblefor the shape and position of the nucleus, was found tobe disorganized, and the microtubules, lost their radialdisposition. After 24 h exposure to modeled low gravityconditions an initial reorganization of the actin and tubu-lin network in J-111 cells was taking place [Meloni et al.,2006]. A similar reorganization of the microtubule net-work was also observed in Jurkat cells exposed to lowgravity conditions [Lewis et al., 1998]. But, despite thefact that the morphology of the microtubule network hada normal aspect, the functional state of the cells did notreturn to normal as the cells did not proliferate. In a cellfree system, the self-assembly of microtubules was foundto be inhibited in low gravity [Papaseit et al., 2000].Vinculin is one of the most prominent membrane-

cytoskeletal proteins in focal adhesion plaques and isinvolved in linkage of integrin adhesion molecules to theactin cytoskeleton. These adhesion sites serve as tractionpoints for impellent forces that push the cell moving for-ward. Vinculin appears to facilitate the assembly of focaladhesion plaques by crosslinking and recruiting its variouspartners [Cox and Huttenlocher, 1998]. Vinculin’s abilityto interact with integrins to the cytoskeleton at the focaladhesion appears to be critical for control of cytoskeletalmechanics, cell spreading, and lamellipodia formation.Thus, vinculin appears to play a key role in shape controlbased on its ability to modulate focal adhesion structureand function [Ezzell et al., 1997; Goldmann and Ingber,2001].In J-111 cells exposed for 24 h to low gravity, we

observed that the vinculin proteins are not evenly spreadbut thickened close to the cell membrane as globular clus-ters. Fewer and smaller focal adhesion plaques can inducea decrease of cell spreading and migration [Pirone et al.,2006].It is interesting to note that the structures of F-actin and

b-tubulin of monocytes of the in-flight 1g samples are simi-lar to those in the GC, despite the fact that these cells havebeen exposed for 55.5 h to low gravity before they have beenloaded on the 1g reference centrifuge in Kubik. Thus thestructures of F-actin and b-tubulin in the J-111 cells havecompletely recovered within 24 h at 1g.Based on our results – alterations in the structures of F-

actin, b-tubulin and vinculin – and those of other authors

it can thus be speculated that the impaired motility of ad-herent monocytes in low gravity on gold particle coatedcover slides might be due to the disruption of the cytos-keletal network. The fact that the monocytes lost theirability to migrate may also be responsible for a hindereddelivery of the costimulatory signal in T cell activation,important for a full activation. Indeed, in several investi-gations we have found that the mitogenic activation ofhuman lymphocytes is suppressed by more than 90% inlow gravity [Cogoli and Cogoli-Greuter, 2007].The cytoskeleton has been described to be the structure

through which the cells sense gravity [Ingber, 1999]. Inmost cells, shape is determined and maintained by cyto-skeleton polymerization forces, weaker in some cell typesthan in others [Todd, 1989] and extension of microtu-bules to the cell membrane [Osborn and Weber, 1976] tomaintain mostly uniform cell surface tension. In the ab-sence of gravity, very subtle cell volume changes mayresult from hydrostatic pressure shifts, potentially causingdisjunction between the membrane and critical cytos-keletal elements [Todd, 1989]. Mechanical changes maybe transduced into biochemical responses through thecytoskeletal scaffolding within the cell [Ingber, 1997]. Ifcytoskeletal structures as F-actin and microtubules butalso the intermediate filaments of vimentin are disrupted,molecular transport by cytoskeletal elements would beaffected and cell surface, receptor-dependent signal trans-duction reactions could not occur. Besides the changes inthe structures of F-actin and microtubules in monocytes,lymphocytes and other cells, reported by us and otherauthors we also have found significant changes in thestructure of vimentin in Jurkat cells [Cogoli-Greuter et al.,1998]. These alterations were observed already after an ex-posure of 30 sec in low gravity and consisted in the for-mation of thick bundles compared to the fine network inthe 1g control.In the present investigation we have found that the mo-

tility of adherent monocytes is remarkably reduced in lowgravity. Furthermore the cytoskeletal structures of F-actin,b-tubulin and vinculin are severely damaged. We thusspeculate that the disruption of the three different cytos-keletal elements is the main reason for the loss of the abil-ity of the J-111 cells to migrate. A disruption of thedifferent cytoskeletal structures in monocytes may alsohave an influence on the differentiation of monocytes inlow gravity and hence affect their role in the innateimmune system.Different cells of the innate immune system including

monocytes are known to be sensitive to gravity changes.In blood samples of astronauts returning from spacechanges in the number of monocytes and natural killercells were observed depending on mission duration. Aftera 9-day mission, monocytes were increased while naturalkiller cells were decreased. However, monocytes weredecreased after the 16-day missions whereas no change

n 134 Meloni et al. CYTOSKELETON

occurred in natural killer cells [Stowe et al., 2003]. Fur-thermore, an increase in neutrophil granulocytes wasfound, but their phagocytic and oxidative functions signif-icantly decreased [Kaur et al., 2005]. In low gravity,monocytes lost their capability of secreting IL-1 [Cogoli,1993] and expressing IL-2 receptor [Hashemi et al.,1999]. Space flight experiments revealed that the distribu-tion, cellular quantity and kinetics of translocation ofPKC – a key protein controlling growth and differentia-tion of monocytes into macrophages – are altered inU937 monocytes in low gravity [Schmitt et al., 1996;Hatton et al., 1999, 2002]. Recently, the examination ofgene expression of monocytes under low gravity demon-strated significant changes in gene induction associatedwith differentiation of monocytes into macrophages[Hughes-Fulford et al., 2008]. On the other hand, exten-sive studies in modeled and low gravity in space revealedthat the cytotoxic effects of natural killer cells were notaffected by gravity changes [Buravkova et al., 2007].There are severe limitations in the number and type of

experiments that can be conducted in low gravity in space.Therefore we have performed many studies with immunecells – lymphocytes and monocytes – in the RPM. TheRPM is a device creating similar gravity conditions asobtained in space by randomizing the gravity vector.When we compare the data of the present investigation inspace with the results obtained with the same cells – J-111 monocytes – in modeled low gravity [Meloni et al.,2006] we can conclude that this is a suitable tool for grav-ity research on ground. Furthermore we also found a verygood concordance of the results obtained with humanlymphocytes in space as well as on ground in modeledlow gravity [Cogoli and Cogoli-Greuter, 2007].Qualitatively, the results from space-flown immune cells

in cultures, and cells exposed to modeled low gravity, areoften similar to the effects space flight exerts on functionalaspects of immune cells obtained from cosmonauts/astro-nauts after their return to Earth. The extensive researchon immune cells in culture under different gravity condi-tions contributes significantly to the understanding ofgrowth responsiveness during space flight and may help inpredicting potential compromise to immune functions inhumans during long duration missions.

Acknowledgment

This project was supported by grants of the Italian SpaceAgency (Grant number: MoMa I/014/06/0) and thePRODEX program of the European Space Agency (ESA;Grant number: 98-HEDS-02-12). The authors wish tothank the hardware developer Daniele Piazza for his con-tinuous support, the teams of ESA and RSC Energia forthe integration of experiment MIA in the ESA KubikBIO#1 mission, the ESA Moscow office for support inMoscow and Baikonour, the ESA user centers MUSC in

Cologne and BIOTESC in Zurich for their support dur-ing the mission, Ludmilla Buravkova and her assistantfrom the Institute of Biomedical Problems in Moscow forhosting and supporting us for the ground control experi-ment. Special thanks go to cosmonaut Pavel Vinogradowwho performed the experiment on the ISS with great chal-lenge. Furthermore we would like to acknowledge A.G.Campus for his invaluable technical assistance.

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