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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 5, 2001 as doi:10.1096/fj.00-0527fje. |
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* AIPC Lab., Université Paris 7, IUH, Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75475 Paris cedex 10, France;
Dept. of Internal Medicine I, University Hospital, Ulm, Germany, and IMAGENIUM, 33 rue St Roch, 75001 Paris, France;
Pharmacologie Lab., IUH, Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75475 Paris cedex 10, France;
U353 INSERM, IUH, Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75475 Paris cedex 10, France;
GSBMS, Université Paul Sabatier, Toulouse, France; and
# U430 INSERM, Hôpital Broussais, Paris, France
2Correspondence: AIPC Lab, UP7, Institut Universitaire d’Hématologie, Hôpital Saint Louis, 1, avenue Claude Vellefaux, 75475 Paris cedex 10, France. E-mail: jvassy{at}chu-stlouis.fr
SPECIFIC AIM
This study aims to investigate the role of gravity in signal transduction across the cytoskeleton to the nucleus. According to our hypothesis, gravity was considered as an external force and, depending on whether it was applied (1g ground controls or 1g centrifuge in-flight controls) or not (weightlessness), the spatial organization of the cytoskeleton—and consequently, the cell physiology—would be modified. We postulate that the integration of both biochemical and mechanical signals might induce specific cytokeratin-architecture patterns, characterized by quantitative image-analysis features, previously described by our group.
PRINCIPAL FINDINGS
1. In µg, cell spreading was reduced and the number of Ki-67
positive cells (cycling cells) was increased
The experiment was performed in the IBIS instrument (Instrument de
Biologie Spatiale) developed by the CNES (French Centre National
d’Etudes Spatiales). IBIS contains two sets of cassettes: one was kept
in weightlessness (10–5 residual gravity,
µg), while the other was kept in a 1g
centrifuge (1g in-flight control). Human breast cancer cells
MCF-7, flown in space in a photon capsule, were fixed in 1.5%
paraformaldehyde and 0.1% glutaraldehyde after 1.5
(t0), 22 (t1), and 48 h (t2) in orbit. Time t0,
just before the 1g in-flight centrifuge started, was selected to study
the effects of launching stress; for example, vibrations and
acceleration. Cells subjected to weightlessness (abbreviated by
µg) were compared with 1g in-flight and ground
controls.
Double fluorescent labeling was used to detect Ki-67 (nuclear antigen, found in cycling cells) and microfilaments (Texas Red-phalloidin, TR-phalloidin) simultaneously. TR-phalloidin staining was used to assess cell spreading and to count the total number of cells (Ki-67 positive and negative one) in each image. Ki-67 positive cells were recognizable by their nuclear and nucleolar staining (FITC) in interphase cells (most of them) or by chromosome staining in mitotic cells. Noncycling cells at the time of fixation were Ki-67 negative.
At time t2 (48 h after launching), cells were more fully spread in 1g in-flight control than in µg. The fraction of cycling MCF-7 cells was calculated as the ratio of the number of Ki-67 positive cells/total number of cells. When comparing 1g in-flight controls and µg, cycling cells were significantly more numerous in µg than in 1g at time t2 (P=0.011) and t1 (P=0.046).
2. Mitosis duration was increased
The number of cells in a specific phase of the cell cycle is
proportional to the duration of that phase. Here, the duration of
mitosis was estimated by the ratio of the number of mitotic
cells/number of cycling cells. Whereas mitosis duration in
1g at both t1 and
t2 were similar to t0,
µg significantly prolonged it (P=0.011). As
several cells in anaphase were still observed, we can hypothesize that
the cell cycle would be blocked only partially in
G2M. Finally, MCF-7 cell proliferation was
reduced in µg.
3. Some MCF-7 clusters showed altered microtubules (MT)
Compared with ground controls, in which MT were
uniformly labeled on all the coverslips, some clusters of MCF-7 showed
modified MT, even at time t0, 1.5 h after
the launching stress. At time t2 (48 h after
launching), cells seemed to have reestablished normal MT in
1g in-flight control (Fig. 1a
, b
, c
), but not in µg
(Fig. 1d
, e
, , f
, , g
, h
). Instead of long, strongly labeled MT
radiating throughout the cytoplasm, only a few filaments could be
distinguished against the strong (gray) background. At the same time,
labeled lamellipodia were observed in these cells (Fig. 1d
).
This more or less diffuse labeling could correspond to either labeled
free tubulin subunits or numerous but very short MT, as visualized in
high-magnification images (Fig. 1g
, h
). Differences were
clearly seen in isolated cells (Fig. 1c
vs. Fig. 1f
). However, some cell clusters contained diffusely
labeled MT in µg and in 1g in-flight controls (Fig. 1b
). Furthermore, labeling patterns of neighboring cells
showed diffuse or well polymerized MT (Fig. 1h
, left),
whereas MF were well organized in both cell types, as demonstrated by
TR-phalloidin staining (Fig. 1h
, right). Thus, MT
alterations could not be attributed to technically faulty cell
fixation. At high magnification (Fig. 1g
), fluorescent
spots, close to the limit of the light microscope resolution and
sometimes aligned over about 2 µm, were seen at the cell periphery,
where the cytoplasm is very thin (2 or 3 µm thick). These
observations could suggest the existence of short MT, with no
preferential orientation, and some seemed to be perpendicular to the
substratum. This finding is in contrast to the preferential orientation
toward the cell periphery and parallel to the coverslip, which is
obvious in the ground control and 1g in-flight control (Fig. 1c
) at the cell boundary.
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4. The regular pattern of perinuclear cytokeratin network
was significantly altered after two days of culture in µg
Cytokeratin networks were clearly observed in the
1g in-flight control and in µg, at all the
times studied (t0, t1,
t2). Sometimes, however, unusual patterns were
seen in µg at time t2. In the ground
control, cytokeratin networks presented characteristic patterns
depending on their intracellular localization. In particular, the
network around the nucleus generated a constant pattern, previously
described as ‘alveolar’ (Portet et al., 1999). The meshes of the
perinuclear network were often looser in µg than in
1g in-flight control, mainly at time
t2. Quantitative image analysis of the meshes in
these networks showed that this pattern was significantly altered after
48 h of culture in µg
(t21g vs.
t2µg, P=0.004).
5. Chromatin distribution in interphase nuclei was significantly
modified after two days of µg
DNA staining was performed by using the fluorescent antibiotic
chromomycin A3, specific to G-C-rich regions.
Quantitative image analysis showed that chromatin distribution
(excluding nucleoli) was denser in 1g in-flight control than
in µg, mainly after 48 h of culture
(P=0.001).
CONCLUSIONS
If physical characteristics of the cell environment guide cell physiology (proliferation or differentiation), weightlessness could alter the relationships between cell structure and function. Actually, in our weightlessness experiments, MCF-7 human mammary carcinoma cell line presented several functional and structural alterations.
First, immunofluorescent labeling of Ki-67 confirmed that cell cycling was altered under weightlessness conditions. Our results also showed that the increase of cycling cells in weightlessness is due to the prolongation of mitosis. Our observations agree with the Jurkat cell line results obtained by Lewis et al. (1998), who used flow cytometry to demonstrate a blockade of the cell cycle in G2M, or in S and G2+M phases of the cell cycle, as demonstrated by Cogoli-Greuter et al. (1996).
The longer MCF-7 mitosis duration could be explained by the alteration of MT in weightlessness. MT alterations were observed after launching, and their normal organization was reestablished 48 h later in 1g in-flight controls. These findings suggest that MT dynamics were affected by weightlessness, in agreement with in vitro experiments reported by Tabony and co-workers (Papaseit et al., 2000). Self-organization of MT into stationary macroscopic patterns is gravity dependent, and the patterns correspond to different MT orientations. The resulting patterns reflect what happens during a critical period of 6 min after the onset of MT assembly. In our experiment, cells were subjected to 5g for 5 min (launching) then to weightlessness for 1.5 h before the 1g centrifuge was started (t0). As MT turnover is 10-fold faster during mitosis compared with interphase, the faster MT turnover corresponds to density fluctuations of free tubulin subunits and represents a window during which the system can interact with gravity. In weightlessness, the existence of short MT, which had lost their preferential orientation, could be in agreement with Tabony’s in vitro experiments.
In addition, MCF-7 cells usually contain more than one MTOCs and micronuclei, which would correspond to the centrosome amplification, defective G2M cell-cycle checkpoint, and micronuclei previously described in breast cancer with BRCA1 and BRCA2 mutants. Thus, the presence of multiple points of MT nucleation would render MCF-7 cells particularly sensitive to weightlessness.
Several patterns of cytokeratin networks have been described in MCF-7 cells, depending on their intracellular localization. Our hypothesis was that architectural variations would depend on local tension or forces, in agreement with the tensegrity paradigm of Ingber (1993, 1999). The perinuclear network pattern is particularly constant on Earth. It was significantly more loosely woven, especially after 48 h of culture in weightlessness. The latter findings, in association with the less extensive cell spreading, would be consistent with the basic predictions of cellular tensegrity, particularly the role of perinuclear cytokeratin network in mediating the transfer of mechanical signals from the cell surface to the nucleus.
According to this tensegrity hypothesis, weightlessness might induce modifications in nuclear architecture and gene expression. In our experiment, quantitative analysis of chromatin structure showed that, after 48 h, weightlessness had modified DNA distribution in interphase cells. Chromatin modifications may have two effects on cell physiology: 1) gene expression and cell differentiation, subsequent to modification of nuclear organization (7, 9), and 2) cell proliferation, following decreased mechanical signaling mediated by the cytokeratin network.
In conclusion, our observations support the hypothesis that the two
mechanisms (reaction-diffusion processes and tensegrity) could be
simultaneously affected by weightlessness. We summarized in Fig. 2
several mechanisms that could be involved in the effect of
weightlessness on cell physiology.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0527fje ; to cite this
article, use (February 5, 2001) FASEB J. 10.1096/fj.00-0527fje 
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-tubulin in MCF-7 cells.
Scale bar is 10 µm. a–c) t21g (1g in-flight
control, cell fixation 48 h after launching). d–h)
t2 µg (µg, cell fixation 48 h after launching).
a–g) 