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Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions ¹

Laboratoire de Biologie et de Biochimie du Tissu Osseux-Equipe Mixte INSERM E9901, Université Jean Monnet, Saint-Etienne. Laboratoire TIMC, UMR 5512, Institut Albert Bonniot, Université Joseph Fourier, Grenoble, France. ¹

²Correspondence: Laboratoire de Biologie et de Biochimie du Tissu Osseux-Equipe Mixte INSERM E9901, Université Jean Monnet, Saint-Etienne, 15 rue Ambroise Paré, F-42023 Saint-Etienne Cedex 2, Grenoble, France. E-mail: guignand{at}univ-st-etienne.fr

SPECIFIC AIMS

We addressed the hypothesis that exposure to microgravity modified the cytoskeletal dynamics in osteoblasts. We monitored morphological and topographical cytoskeletal structures of cell adhesion in ROS 17/2.8 cells during several days of real microgravity and analyzed the relationships between cell cycle and adhesion pattern after 4 days of flight.

PRINCIPAL FINDINGS

1 Quantitative cell adhesion parameters: microgravity acts as a destabilizing agent
Adhesion plaques, which link intracellular cytoskeleton to extracellular matrix and trigger a myriad of intracellular signals, have a recognized role in mechanotransduction both in mechanical stress models and in the case of gravitational stress such as that exerted by parabolic flights. Figure 1 shows characteristic images of all the parameters studied and illustrates differences between 1 g and micro-g in flight cells. The present study is the first to provide quantitative data on cell adhesion after microgravity exposure. We collected two types of cell adhesion information. The first was related specifically to integrin-mediated adhesion and was based on vinculin and phosphotyrosine (PY-20 antibody) imaging with confocal microscopy at focal adhesion sites (=spots). The second was not specific and represented physical contact zones at cell/substratum interface as imaged by TIRFM (=plaques). The image analysis software provided 18 morphometric features describing cellular area, shape, and proportions of vinculin (and PY-20) spots as well as 6 topographical features describing the distribution of vinculin and the relative overlap of spots and plaques. Stepwise factorial discriminant analysis indicated the discriminant power of each parameters in all the groups at every time point. As early as 24 h of flight, mean area of vinculin spots (MS) was significantly decreased compared with centrifuge or ground controls. After 2 days in space, similar results were seen for vinculin relative area (RS), overlap of vinculin spots with plaques (OSP), and average distance of vinculin spots from the cell edge. After 2 days of flight, PY-20 staining indicated that the phosphotyrosine-positive spot number and phosphotyrosine-related area were also significantly decreased (-50%) in the flight group vs. both centrifuge and ground groups. The ratio of PY-20 to vinculin spots was also dramatically reduced in microgravity, suggesting that vinculin left the focal contacts and transduction activity of this signaling molecule was reduced. After 4 days of flight, all morphological and topographical adhesion parameters remained low. Distribution of vinculin spots was restricted mainly to the cell periphery and at the nucleus level (loss of intermediate contacts) as shown by reduced average distance from cell edge to spots. The longer the flight, the greater the differences. Parameters derived from plaques (TIRFM) were not significantly altered by space conditions. Lack of overlap between plaques and vinculin spots (OSP) appeared early and continued to deteriorate until the 4th day of flight.

View larger version (83K): [in this window] [in a new window]   Figure 1. Observation by phase contrast microscopy of cell morphology after 4 days of culture. A) Centrifuge in flight controls. B) Flight culture. Note that after 4 days, a large proportion of flown cells are retracted or round compared with the well-spread morphology of centrifuge. Confocal microscopy analysis of the 2-day centrifuged group of cells (C, E, G) and microgravity-exposed cells (D, F, H). C) Centrifuge controls stained for F-actin. D) Centrifuge stained for F-actin; note that F-actin is restricted to cell edge (filopodia-like extensions) and rarely is organized in stress fibers vs. the centrifuge well-organized cytoskeleton. E, F) Vinculin patterns indicating an important diffusion of vinculin out of focal contacts in flight cells. G, H) Arrows indicate G1, M, and S/G2 cell cycle phases discriminated by Ki-67 staining topography. Note that flight cells are cycling normally. Bars: 50 µm (A, B); 15 µm (C–H).

These results indicate that dynamic disassembly of vinculin was not associated with parallel changes in plaque area. This was unexpected, as close contact and focal adhesions are usually related structures formed from a common hierarchy of molecular interactions. The mechanical environment is known to be crucial for the maintenance of cell integrity, as the cytoskeleton-generated tension forces that equilibrate external tension are mainly provided by cell–matrix and cell–cell interactions, pressures, and fluid shear stress. This concept, called tensegrity, has led us to the assumption that the absence of external forces (reduced g level, shear stress, and pressure) leads to unbalanced cytoskeletal tension at the beginning of the flight and that longer exposures will lead to a reduction of cytoskeleton-generated tensions. Adhesion to extracellular matrix triggers phosphorylation of a large number of signaling proteins at the focal contact, such as focal adhesion kinase (FAK), p60src, or paxillin, which in turn activate protein kinase C (PKC) and GTPases pathways. Based on the marked decrease of tyrosine phosphorylation at focal adhesion sites in flight cells, we assumed these signaling proteins could be altered.

2 Adhesion cell cycle dependency: postmitotic cell adhesion was specifically affected
ROS 17/2.8 cell cycling (quantified through Ki-67 staining) occurred in microgravity at a rate similar to controls generating the same proportion of cells classified as premitotic (PreM), mitotic (M), and postmitotic (PostM) groups of cells. We tested whether microgravity was able to modify adhesion according to cell cycle phases. Analysis was done after 4 days in microgravity to ensure that we analyzed the second or third generation of ROS 17/2.8 grown in space. Among sorted parameters (by factorial discriminant analysis), Number of spots and MS were the more discriminant parameters. In control cells, progression toward mitosis induced large cell retraction (-100%) associated with an important decrease in number of vinculin spots (-300%). Statistical analysis indicated that microgravity conditions affected more PostM cells than PreM or M cells. Mitotic cells were unaffected by microgravity. In PostM cells, changes in adhesion were characterized by relocation of vinculin and PY20 spots at the cell periphery. Furthermore, large and central spots were lost and F-actin was disorganized, showing a cortical pattern.

In centrifuge and ground control groups, the presence of large central spots was indicative of normal spreading and stability of focal adhesions. A summary of the main results found between adhesion and cell cycle phase in centrifuge and flight samples is schematized in Fig. 2 .

View larger version (20K): [in this window] [in a new window]   Figure 2. A representative example of binary mask of vinculin spots and normalized distance histogram is presented for each cell cycle phases (pre-M, M: mitotic and post-M cells). Note that postmitotic cells in flight conditions show a smaller cell area and the absence of central spots. This illustrates that cell cycle progression occurred in flight and suggests that spreading of postmitotic ROS 17/2.8 cells is modified by microgravity. It appeared that in flight conditions (right column), cells are more irregular in shape (higher shape factor indexes) and present distance histogram of spots restricted in the periphery of the cell (cell edge peak) explained by disappearance of stable, central contacts leading to disorganization of ROS cytoskeleton characterized by absence of actin stress fibers.

This type of vinculin distribution in peripheral focal complexes has already been described when Rho activity is inhibited. Peripheral complexes were considered to be precursors of focal adhesion plaques. We can therefore speculate that focal adhesion spots generated in microgravity are less mature than those established in controls. Maturation of focal contacts is a complex process, depending on the dynamics and stability of actin cables, and is under the control of Rho members of GTPases. Loss of F-actin fibers observed under flight conditions could be explained by loss of central adhesion spots required for the formation of stress actin fibers. Under microgravity conditions, cell adhesion could be restricted to immature small spots.

3 Proliferation and differentiation indices were not affected by space-related conditions
Both protein content and direct counting of cells were identical between controls, centrifuge, and flight cells all over the culture periods in both missions. Osteoblastic differentiation parameters—i.e., alkaline phosphatase activity, carboxyl-terminal type I procollagen propeptide (P1CP) and osteocalcin production—were similar between the three conditions, indicating that ROS 17/2.8 phenotype was not specifically affected during spaceflight conditions.

CONCLUSIONS

ROS 17/2.8 cells retained their capacity to proliferate and their osteoblastic phenotype after a 6-day microgravity exposure despite alterations in cell adhesion. Quantification of focal adhesion parameters demonstrated that microgravity acts on ROS17/2.8 cells by disorganizing cytoskeletal actin and vinculin. The kinetics of the dynamic changes affecting the area and relocation of focal contacts suggested that these structures are not only mechanoreceptors, but also mechanoeffectors. This disorganization was maximal on nonmitotic cells vs. mitotic cells and could be explained by the immaturity of the contacts established in flight (see Fig. 3 ). Rho-GTPase activity or downstream Rho effectors could be affected by changes in the mechanical environment experienced under microgravity conditions and might explain our results. Additional experiments are needed to precisely study GTPase activities under microgravity conditions. This experiment suggested that space-related conditions drive cytoskeletal restructuring, which might control signaling cascades and thereby govern the cellular response to other stimuli such as hormones or growth factors.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram. OB, osteoblasts; FC, focal contacts; a, F-actin fibers; n, nucleus.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0837fje ; to cite this article, use FASEB J. (July 24, 2001) 10.1096/fj.00-0837fje

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This Article Full Text (PDF) All Versions of this Article: 15/11/2036 00-0837fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GUIGNANDON, A. Articles by VICO, L. Search for Related Content PubMed PubMed Citation Articles by GUIGNANDON, A. Articles by VICO, L.