Acta Astronautica 56 (2005) 613–621

www.elsevier.com/locate/actaastro

Application ofGFP technique for cytoskeleton visualizationonboard the International SpaceStation�

E.L. Kordyuma,∗, G.V. Shevchenkoa, A.I.Yemetsb, A.I. Nyporkob,Ya.B. BlumebaInstitute of Botany, National Academy of Sciences of Ukraine, 2, Tereshchenkivska St., 01004 Kyiv, UkrainebInstitute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv, Ukraine

Received 25 April 2003; received in revised form 13 September 2004; accepted 19 October 2004

Abstract

Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensingand trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different mod-ifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles,they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocyto-chemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells isneeded for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletalvisualization onboard the ISS, namely, application of green fluorescent protein (GFP) fromAequorea victoria, which hasthe unique properties as a marker for protein localization in vivo. The creation of chimerical protein–GFP gene constructs,obtaining the transformed plant cells possessed protein–GFP in their cytoskeletal composition will allow receiving a simpleand efficient model for screening of the cytoskeleton functional status in microgravity.© 2004 Elsevier Ltd. All rights reserved.

1. Introduction

The investigations in space and gravitational bi-ology performed during last 25 years have demon-strated the significant rearrangements in the cell struc-tural organization, metabolism, and basic processes in

� Based on paper IAC-02-T.6.03 presented at the 53rd Inter-national Astronautical Congress, 10–19 October 2002, Houston,USA. Supported by Grants STCU-NN-01(R) INTAS 03 51 6459.

∗ Corresponding author. Tel./fax.: +38442123236.E-mail address:cell@svitonline.com(E.L. Kordyum).

0094-5765/$ - see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.actaastro.2004.10.006

prokaryotes and eukaryotes in situ and in vitro underthe influence of microgravity[1–4] that underlined afundamental discovery of cell gravisensitivity. Threemain, closely interconnected questions aroused on thebasis of the discovery of cell gravisensitivity, namely:(1) what are the mechanisms of the influence of micro-gravity at the cellular level, (2) how cells of differenttypes perceive gravity, and (3) what possibilities andmechanisms of cell adaptation to this factor causingthe changes in such physical parameters as sedimenta-tion, convection, capillarity, hydrostatic pressure, andsurface tension. In search of an answer to such the

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questions, significant attention is paid to cytoskeletonwhich is assumed to be an indicator of cell functionsdetermining organism gravisensitivity. One of the use-ful models for investigation of cell gravisensitivity arehigher plants where the sites of gravity perception andreaction are spatially separated. From this point ofview, the localization of cytoskeleton elements at thedifferent stages of plant development with special em-phasis on cytoskeleton dynamics during developmentof root graviperceptive and graviresponsive regionsare of a special interest. In the given paper, the dataon cytoskeleton organization and dynamics in plantcells under the influence of altered gravity—real mi-crogravity in space flight and clinorotation, that par-tially reproduces the biological effects of micrograv-ity, are considered and certain approaches to futurecytoskeleton investigations in spaceflight and ground-based experiments, including the application of GFPtechnique, are discussed.

2. Cytoskeleton and cell gravisensitivity

2.1. Concepts and hypotheses

According to the first concept of positional home-ostasis[5], a stable position and optimal orientation ofcells in the gravitational field are defined by the stateof the mechanical straight of intracellular cytoskele-tal elements (microtubules and microfilaments) andthe integrity of cell membranes. Cytoskeleton, its me-chanical characteristics, functional activity, biochemi-cal properties, and ultrastructural organization are alsoconsidered as a cell integral not-specialized gravire-ceptor[6]. Actin filaments of cytoskeleton are consid-ered to be a key component in cell gravisensitivity ac-cording to the hypothesis of protoplast pressure[7]. Asignificant role in the stability of cell spatial–temporalorganization and its gravisensing is attributed to thecytoskeleton in other concepts of cell gravisensitivity,namely: static stimulation[8], passive gravistimula-tion [9], and restrained gravisensitivity[10]. Accord-ing to the hypothesis of putative tensegrity[11], thecellular integrity is based on tensional forces origi-nating from the actomyosin complex and are resistedby microtubules and the cytoplasmic membrane sup-ported by the extracellular matrix. Cytoskeleton func-tions include mitotic division and cytokinesis, intra-

cellular transport, endo and exocytosis, and single celland tissue motility; these are all the activities that arepotentially gravity sensitive[12]. The intracellular cy-toskeleton, the extracellular matrix, and the cytoplas-mic membrane is assumed to represent compartmentsof sufficient macromolecular organization to be sensi-tive to gravity-induced phenomena[2]. Cytoskeletonand extracellular matrix are indispensable for cellu-lar and developmental processes which are directly orindirectly linked through the cytoplasmic membraneas an intermediary between them. Cellular receptorsin the wall/extracellular matrix probably bind to in-tegrins that span the cytoplasmic membrane and thenconnect to the cytoskeleton through other proteins.In animal cells, microfilaments and membrane inte-gral proteins are linked in the specific domains of thecytoplasmic membrane with such proteins as tensin,vinculin or �-actinin. Fibronectin and intronectin arejoined to these domains from the side of extracellularmatrix[13,14]. According to the hypothesis of gravita-tional decompensation[3], during reduction or the ab-sence of hydrostatic pressure, a change in the surfacetension of the cytoplasmic membrane can trigger therearrangements of its physical and chemical proper-ties. In addition, the events termed “piezoeffects” cancomplicate the tension state of the membrane surface.Briefly, they describe a mechanical tension at the levelof single molecules that appear and disappear in thecytoplasmic membrane due to cytoplasmic streaminginvolving cytoskeleton elements. One of the key path-ways in transfer of a mechanical signal from the mem-brane surface is supposed to be the rearrangements inintegrin—vinculin—internal cytoskeleton linkages.Based on experimental data showing a strong effect

of microgravity on cell metabolism, a concept was putforward that proliferating and actively metabolizingcells are the most sensitive to the influence of alteredgravity. Therefore, it is proposed to distinguish be-tween cell gravisensing and cell graviperception: thefirst is related to cell structure and metabolism stabil-ity in the gravitational field and their changes in mi-crogravity (a discovery of gravisensitivity of cells notspecialized to gravity perception was done with theresults of space experiments). The second one is re-lated to actively using gravitational stimulus by cells,especially specialized for gravity perception. It allowsrealization of plant normal space orientation, growth,and vital activity (gravitropism, gravitaxis)[15].

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2.2. Cell graviperception

The structure of graviperceptive cells is diverse, butgravisensors are well known. These are statoliths ofdifferent types that change their position in the direc-tion of the gravity vector and thus initiate the nextsteps of the gravitational response. The structural andfunctional organization of graviperceptive cells is de-termined genetically[4,16–22]. The most useful mod-els for the investigations of cell gravisensitivity inspaceflight and clinostat experiments are gravipercep-tive cells with statoliths, e.g. root cap statocytes, algaChara rhizoids, apical cells of moss protonema.

2.2.1. Tip-growing plant cellsThe apical and subapical zones ofChara rhizoids

contain thin bundles of microfilaments and the basalzone contains thicker ones. The apical part containsstatoliths—compartment filled with crystallites of bar-ium sulfate—and is gravitropically responsive[8]. Itwas concluded that the arrangement of microtubulesis essential for polar cytoplasmic zonation and polarorganization of actin cytoskeleton but is not involvedin the primary events of gravitropism[23]. The actinmicrofilaments are involved not only in cytoplasmicstreaming and the maintenance of functional cell po-larity and tip growth but also in gravitropism throughthe positioning transport, and sedimentation of sta-toliths.An apical cell of moss protonema displays a certain

internal structural polarity. In a cell beginning fromthe apex, there are the following zones: the most distalapical zone, which does not contain plastids; the zonewith numerous chloroplasts, which extends from theapical zone to a nucleus, and the basal, proximal zone,which is very vacuolized[24]. Amylochloroplasts inthe apical cell ofPottia intermediachloronema canhave a different shapes—rounded (in the distal partof a cell) and spindle-like (in the proximal part of acell). Rounded amylochloroplasts contain large starchgrains[25] and they are believed to play the role ofstatoliths[25–27]. The main role of microtubules inprotonema gravisensitivity is considered[28] based onthe data on their enrichment during graviresponse inthe distal apical zone, which does not contain plastids.Certain reorganization of microtubules under gravis-timulation and in altered gravity in protonema api-

cal cells allowed to assume their contribution in bothgraviperception and plastid reorientation inmicrograv-ity [29].

2.2.2. Root cap statocytesStatocytes as highly specialized graviperceptive

cells are characterized by the structural polarity shownby the position of a nucleus in the proximal part of thecell and statoliths in the distal part of the endoplas-mic reticulum membranes. Amyloplasts performing astatolithic function sediment in the distal part of thestatocytes in the direction of a gravitational vector[16] and rather spread over the entire volume of thestatocyte cytoplasm in microgravity[3,30]. The polararrangement of organelles is achieved and maintainedby means of the cytoskeleton[8,31,32]. These as-sumptions have been confirmed by the experimentsperformed in microgravity and under clinostating us-ing the actin-disrupting drug cytochalasin D[30,33].A conclusion was made that on Earth the position ofstatoliths in both rhizoids and statocytes depends onthe balance of two forces: the gravitational force andthe counteracting force mediated by actin microfil-aments. Myosin-related proteins were also found inplastid membranes[10]. The presence of�-integrin-like polypeptide in the inner membrane of amyloplasts[34] suggests similarity to functional mechanosensingmolecules in animals. It is supposed that some differ-ences in the actin cytoskeleton organization betweenroot and stem statocytes may determine the differ-ent degree of root and stem gravisensitivity[22]. Itwas shown that ARG1 (altered response to gravity)locus inArabidopsis thalianacan affect gravitropismof roots and hypocotyls. ARG1 locus encodes DnaJ-like protein containing a coil-like region homologuesto the region of proteins, which interact with thecytoskeleton[35].

2.3. Graviresponse in the root elongation zone

During formation of the root bending as the lastphase of a gravitropic reaction, the most essentialchanges of microtubule orientation occur in the epi-dermis and cortex cells[36]. There is an assumptionon close interaction between microtubule orientationand the auxin concentration, i.e. the orientation of mi-crotubules is a physiological indicator of the effec-tive internal auxin concentration on both sides of a

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root during graviresponse[37]. At the same time, itis supposed that microtubule reorientation is causedmechanically under epidermal cell growth by elonga-tion. This hypothesis explains why not only auxin butthe other growth factors affect microtubule orienta-tion, taking into account that the transverse arrange-ment of microtubules promotes cell growth, the longi-tudinal ones inhibits it. Recently, it was shown somedisturbances of microtubule organization in the cellsof Beta vulgarisroot epidermis and cortex under cli-nostating[38]. In the control, microtubules are mainlyoriented perpendicular to the root longitudinal axis,under clinostating, their orientation became less regu-late, and the essential deflection is observed. Functionsof the actin cytoskeleton in root gravitropic bendingis yet less investigated, and it is assumed that an actinnetwork does not directly participate in graviresponseformation[10].

2.4. Cell gravisensing

In the most cell types not specialized for perceptionof gravity, the primary sensors are not clearly defined.It was shown the participation of actin microfilamentsin gravisensing of plant cells not specialized to gravityperception, e.g. tip-growing root hairs[39]. There isthe actin concentration in the tip of a root hair[40] thatis considered as its direct participation in tip growth.At the earliest stage of hair formation, rhizodermaloutgrowths change their orientation under clinostatingin comparison with control ones (Figs.1 d and e). Ahair orientation restores after the beginning of activetip growth (Figs.1f and g) that is accompanied withthe rearrangements of actin microfilaments in the apexresulting in displacement of an exocytosis point at theapical cytoplasmic membrane. Based on the data on adisappearance of two actin izoforms in wheat meso-phyll protoplasts after 6min of the microgravity actionfrom four revealed before the experiment, the pres-ence of specific actin izoforms responsible for certainfunctions related to gravisensing is supposed[41]. Atleast, a dependence of microtubule self-organization invitro on the gravitational force was recently revealed[42]. Flight results also suggest that polymerization oftubulin into microtubules, known to be regulated byCa2+ ions [43], may be altered in microgravity[44].In this connection, it is important to pay attention tothe modifications in cytosolic concentration of Ca2+

ions in different plant cells, e.g. algaChlorella cells,moss protonema, root cap statocytes, root hairs, andmicrocallus cells, in microgravity and under clinos-tating [45]. It has been shown that rearrangements ofactin microfilaments in the apex of a growing roothair occur simultaneously with the establisment ofthe apical–basal gradient of Ca2+ ions (Figs.1a–c).Microfilaments localize the exocytosis at the certainsite of apical plasma membrane thus, establishing theplagiotropic growth direction. Under clinostating thegrowth direction of root hairs is affected at the earli-est stages of growth and is restored with the furtherroot maturation. Clinostating increased steepness ofCa2+ gradient through activation of mechanosensitivecalcium channels and increased [Ca2+] alters arrange-ment of apical microfilaments affecting the localiza-tion of exocytosis and changing the growth direction.Plagiotropic growth is restored due to inner strongmechanism of its maintenance, where actin and Ca2+gradient play an essential role[39]. It is also knownthat calciummodulates the activity of many cytoskele-tal proteins, e.g. phospholipid-binding proteins, whichare attached to actin cytoskeleton and the cytoplasmicmembrane[46].

3. Approacher to plant cytoskeleton research

The investigations of the cytoskeleton in plant cellsunder the influence of altered gravity and gravistim-ulation goes slowly due to both general difficultiesin studying the plant cytoskeleton and limited ac-cess to the conditions of space flight. Applicationof rhodamine-phalloidin has its own shortcomingsbecause phalloidin stabilizes microfilament actin[47] and, thus, the F-actin level may be artificiallyincreased. Many recent cytoskeletal researches areconducted by the means of immunohistochemistryproviding the application of antibodies specific tocertain cytoskeleton proteins that allows to reveal theseparate components of a cytoskeletal network. In ad-dition, application of antibodies makes it possible tocreate both the 3D-cytoskeleton organization (light-microscopic level) and reveal more detailed positionof its components together with the visualization ofcytoskeleton element contacts with organelles (elec-tron microscopic level). Different modifications ofthese methods are beneficial for the ground-based ex-

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Fig. 1. Schematic depiction (a–c) and microphotoes of root hair formation at the consequent stages. Abbreviations: N—nucleous,Atlantes—microfilaments, black dots—calcium ions.

periments, but for the performance onboard the spacevehicles they are quite complicated and time consum-ing. In addition, immunocytochemistry provides onlystatic images of the cytoskeleton arrangement in fixedcells while its localization in living cells is neededfor the better understanding of cytoskeletal function.In this connection, we propose a new approach forcytoskeletal visualization on board the ISS, in par-ticular, we use an advanced technology of genetictransformation with reporter gene that encodes thegreen fluorescent protein (GFP) from the jellyfishAe-quorea victoria[48] for plant microtubule orientationand dynamics in specialized living cells. Recently ap-peared this method helped to obtain significant dataon protein localization and dynamics in living cells,including plants.

3.1. GFP method

A number of genetic modifications of GFPmoleculehave produced versions of the protein with improvedspectral properties and stability[49–52]. Additionalmodifications to optimize the codone usage for propermRNA processing in plants[53,54] have further in-creased the utility of this reporter gene. In plants, GFPhas been used successfully to track viral movementproteins[55–58], to localize phragmoplastin[59], andto studyRhizobiumNod factors[60]. In addition, GFPhas been used to localize cytoskeletal elements, in-cluding yeast tubulin, neuronal MAP2C, tau34,Dic-tyosteliumactin, and yeast myosin (reviewed,[61], �-[62] and�- [63] tubulins fromDictyostelium, �- [64]and�-tubulin fromCaenorhabditis elegans[64,65].

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To investigate in vivo plant cytoskeleton few differ-ent applications of GFP technology has been used. AGFP-mouse talin fusion protein labeled plant actin fil-aments in vivo and visualized the actin cytoskeleton ingrowing pollen tubes[66]. To visualize reorientationsof cortical microtubules in living elongating cells, itwas constructed[67] a microtubule reporter gene byfusing the microtubule binding domain (MBD) of themammalian microtubule-associated protein 4 (MAP4)gene with the GFP gene, and transient expression ofthe recombinant protein in epidermal cells ofViciafabawas induced. The reporter protein decorated mi-crotubules in vivo and bound to microtubules in vitro.Confocal microscopy and time-course analysis of la-beled cortical arrays along the outer epidermal wallrevealed the lengthening, shortening, and movementof microtubules; localized microtubule reorientations;and global microtubule reorganizations. Using trans-genic Arabidopsis plants expressing a GFP-MAP4 fu-sion protein, which decorated the microtubular cy-toskeleton, other investigators observed reorientationof microtubules during trichome branching[68].First successful attempt to create transgenic plants

for visualization of GFP labeled microtubules, inwhich GFP-tubulin fusion proteins were expressedand incorporated into microtubules ofArabidopsisthalianawithout interference with normal plant life,has been reported by Ueda et al.[69]. They used tua6gene from Arabidopsis to obtain GFP fused productwith the amino terminus of a-tubulin because moder-ate overexpression of�-tubulin (but not of�-tubulin)is tolerated in yeast cells. The fluorescence of the mi-crotubules was due to the fluorescence of GFP-TUA6that was polymerized into the microtubules. How-ever, GFP-labeled arrays have not been visualizedin dividing cells during these experiments. Differentmitotic microtubular arrays were visualized in a lineof tobacco BY-2 stably expressed GFP-MBD aftertransformation by respective construct under the con-trol of a cooper-inducible promoter from yeast[70].Later transgenic BY-2 cells stably expressing a GFP-tubulin fusion protein (BY-GT16) were produced[71]and used for studying of the mode of cortical MTorganization[71], the origin of cortical microtubules[72], the relationship between the dynamics of vac-uoles and microtubules[73] and the roles of actin-depleted zone and preprophase band in determiningthe division site of higher plant cells[74].

The creation of chimaeric microtubule protein-GFPgene constructs, obtaining the transformed plant cellspossessed protein-GFP in their cytoskeletal composi-tion will allow to receive a simple and efficient modelfor screening of cytoskeleton functional status in mi-crogravity. But it is obviously that microtubule assem-bly and function are especially sensitive to changes inthe tubulin proteins[62]. In retrospect, it should notbe surprising that addition of GFP to the amino ter-minus of �-tubulin could interfere with microtubulenucleation and assembly. It was reported[75] that inthe tubulin dimmer the amino terminus of a-tubulinis located between the two molecules of the dimmer.As the dimmers are assembled in head-to-tail arraysin the protofilaments, the amino terminus of�-tubulinis thus located between adjacent dimmers, a locationthat would be predicted to be important for assemblyof the polymer. In addition, it is generally acceptedthat the�-tubulin end of the dimmer is located at theminus end of the microtubule or protofilament[76].Thus, it is not unreasonable for addition of GFP to theamino terminus of�-tubulin to interfere with micro-tubule nucleation.To realize the idea of producing efficient GFP-

tubulin constructs at present state of art to producea transgenic plants with stable and high expressionof chimaeric protein we plan to use mGFP4 (kindlyprovided by Dr. J. Haseloff) and�- and �-tubulingenes from goosegrass (kindly provided by Prof.W.V. Baird). To search and obtain the most efficientconstructions preliminary 3D-structural computermodeling of chimaeric proteins (GFP-tubulins) withmost optimal configuration of the molecules wasdone. Earlier we were successful in predication devel-opment of 3D models of plant tubulins[77–79]. 3Dmodeling of different variants of GFP introductioninto �- and�-tubulin molecules at N- and C-terminiand at different positions very close to C-terminusallowed to select few most interesting compositionsof chimaeric GFP-tubulin sequences. Few of themshould allow to obtain chimaeric molecules of tubulinwith GFP fragment exposed into lumena of micro-tubules. It is planned additionally to ligate the GFPgene with sequences of�- and�-tubulin genes codingcarboxy terminal sequences, which are variable inall tubulin isotypes (approximately 45–90bp). As thepromoters we could use the hybrid promoter contain-ing cauliflower mosaic virus 35S promoter and the

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maize alcohol dehydrogenase intron 1[80]. To gen-erate stable expressing transgenic plants we proposeto use tranformation technique with�/�-tubulin dou-ble gene construct containing on mutant tubulin gene[81]. After selection we have to examine the efficiencyof function of chimaeric GFP-tubulin(s). It can beachieved by means of studying in vitro the inclusionof chimaeric protein in assembly of microtubules.For observation of cytoskeleton dynamics in trans-

formed living cells it is planned to use a standard flu-orescent microscope with fluorescein isothiocyanatefilter set (HQ480/40 excitation filter, HQ535/50 emis-sion filter, Q505LP dichroic mirror; Chroma Technol-ogy, Brattleboro, VT). No significant autofluorescencewill be seen using this filter combination. Confocalimages of GFP expression could be taken also ona Leica DM IBR inverted laser confocal microscopeusing a standard FITC filter providing excitation at480–490nm and emission at 530–540nm. For the ef-ficient application of the above technique onboard thespecial protocol is working out with the inclusion ofbasic training course for crew members.

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