Cancer Research Center of Russian Federation, 115478 Moscow, Russia
Author for correspondence (e-mail: vasiliev{at}cityline.ru)
Accepted August 13, 2001
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SUMMARY |
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Key words: Fibroblast, Epitheliocyte, Microtubules, Microfilaments, Colcemid, Cytochalasin, Morphometry, Cell shape, Micropatterned adhesiveness.
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INTRODUCTION |
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MATERIALS AND METHODS |
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The cells were grown in Dulbeccos modified Eagles medium (Sigma), supplemented with 10% fetal calf serum (Gibco Biocult, Scotland), at 37°C in a humidified incubator supplied with 5% CO2 in air. The cells were plated at an initial density of 100 cells/mm2 on control substrates or 60 cells/mm2 on the special substrates with narrow linear adhesive strips (see below), placed in 30-mm tissue culture dishes. Owing to low initial cell density, most cells (about 70%) on both substrates remained single after 24 hours, that is, they had no visible cell-cell contacts.
Colcemid (Demecolcine, Sigma; 0.2 µg/ml) or cytochalasin D (Sigma; 0.4 µg/ml) were added to the culture medium 19 hours after cell plating; the cells were incubated with the drugs for five hours before examination.
Substrates
For the usual planar substrate with isotropic adhesive surfaces, we used glass coverslips (Chance Propper Ltd., Smethwich, England). Substrates with narrow linear strips of adhesive surface were prepared as follows. The glass coverslips were coated with a thin non-adhesive layer of the biocompatible hydrogel, poly-2-hydroxyethyl methacrylate (poly HEMA, Polysciences, USA) as described by Folkman and Moscona (Folkman and Moscona, 1978). Stock solution was made by dissolving of 6 g polyHEMA in 50 ml 95% ethanol. The mixture was turned slowly overnight at 37°C and then was centrifuged for 30 minutes at 2500 rpm to remove undissolved particles. This stock was diluted with 95% ethanol to a 101 solution, and was then pipetted onto the coverslip surface. The coverslips were dried on a level bench free of vibrations for 48 hours at 37°C. After the alcohol had evaporated, a thin film of optically clear polymer tightly bonded to the glass surface remained. Then the coverslips were heated at 120°C for two hours. Linear cuts through the coat to the coverslip surface were made with a razor blade. The width of the strips was 15±3 µm.
Differential interference contrast microscopy
The live 24-hour-old cultures were examined by video-enhanced microscopy using a Zeiss Axiophot microscope equipped with differential interference contrast (DIC) optic system with 40x0.7 Pl Fluotar objective and Hamamatsu Newvicon videocamera (Hamamatsu, Middlesex, NJ) For recording of images we used video tape recorder SVT-S3050P (Sony, Japan).
Morphometric analysis of cell shape
The outlines of DIC images of single cells, that is, of the cells without any cell-cell contacts, were used for morphometric analysis. The outlines of the 24-hour-old cells were entered into a PCAT computer by tracing on a digitizing tablet (Summasketch II, Summagraphics, UK). TRACER V1.0 software (Copyright Dr A. Brown) was used for entering and storing the cell outlines and for calculating their shape characteristics (Dunn and Brown, 1986). The four shape characteristics were: maximal cell length, cell area, dispersion and elongation indices.
Cell length was defined as the length of a direct line between two points at the maximal distance on a cell outline. Average values of length of groups of cells were calculated. They are designated in the text as the average length of the cell population.
Dispersion and elongation indices were determined and calculated as described by Dunn and Brown (Dunn and Brown, 1986). The purpose of using elongation and dispersion to describe cell shape is that these have simple transformation properties and hence can detect fundamental transformations of cell shape. Elongation and dispersion describe two different aspects of how a shape differs from a circle. Both measure how much the total mass of the shape extends away from its center of gravity but elongation describes how much this extended mass can be reduced by compressing the shape along its long axis and dispersion describes how much extended mass remains.
Fluorescence microscopy
After 24 hours of culture, cells were washed with phosphate-buffered saline (PBS) containing 0.5 mM CaCl2 and 3 mM MgCl2, fixed in 3% paraformaldehyde in PBS for 10 minutes and permeabilized with 0.1% Triton X-100 for one minute at room temperature. For tubulin staining, the cells were fixed with methanol at 20°C for 10 minutes. F-actin was stained with TRITC-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO). Tubulin was stained with an anti--tubulin mouse monoclonal IgG1 (clone DM1-A, Sigma). Paxillin was stained with an anti-paxillin mouse monoclonal antibody (Transduction Laboratories). For secondary antibodies, we used TRITC-conjugated goat anti-mouse IgG (Chemicon) and Oregon Green 488-conjugated goat anti-mouse IgG (Molecular Probes, USA). After several rinsings in PBS, preparations were mounted in buffered polyvinyl alcohol (Lennett, 1978). For fluorescence microscopy, an Aristoplan (Leitz, Germany) microscope equipped with epifluorescence illumination and a 50x1.0 Pl Fluotar water-immersion objective was used.
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RESULTS |
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In order to find out more about the factors controlling fibroblast length, we tested the effects of two drugs affecting cytoskeletal systems: colcemid, which depolymerizes microtubules; and cytochalasin D, which destroys actin structures. Control tubulin staining showed that colcemid-treated fibroblasts had no cytoplasmic microtubules. These experiments showed that colcemid-treated fibroblasts had significantly decreased average cell length compared with untreated cells both on the standard substrates and on the strips (Table 1; Fig. 4b,c).
Cytochalasin-treated fibroblasts extended narrow cytoplasmic processes filled with microtubules and vimentin filaments; they had no lamellae at the ends of these processes. These cells had significantly increased average maximal lengths compared with control cells on both substrates (Table 1; Fig. 4b,d).
In a special experiment, M19 fibroblasts cultivated for 24 hours on the strips were re-seeded onto the standard substrate and examined after another 24 hours. These experiments showed that alterations caused by cultivation on the strips (area, elongation and dispersion) were reversible once transferred to the standard substrate (Table 3).
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Additional experiments with MDCK epitheliocytes showed that morphometric changes observed on the strips were fully reversible: the cells re-seeded from the strips on the plane after 24 hours restored all the average parameters characteristic of epitheliocytes on this substrate (Table 3).
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DISCUSSION |
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In our experiments we assessed only the projection of cell shape on the substrate plane in two dimensions; the third dimension (cell height) was not measured. Possibly, the height was increased when the cell was squeezed on the strip. Further experiments are needed to reveal these possible changes. In any case, it can be concluded from our experiments that the average length of fibroblasts remains relatively constant regardless of other shape parameters on the strip. Of course, only the average length characteristic of the whole population is preserved individual cells may oscillate around the average in the course of their life history. For instance, moving fibroblasts detach and contract their tail processes from time to time, but later their anterior lamella spreads forward restoring their length (Chen, 1979; Dunn and Zicha, 1995). Another example of length oscillation is cell rounding during mitosis. However, mitotic cells at any given time form a very small part of fibroblastic populations in our cultures, so that they could not significantly influence the shape.
Our data show that there are two distinct factors controlling the degree of spreading of elongating fibroblast: factors controlling spreading in the direction of cell length and factors controlling spreading in other directions. We will designate them as longitudinal and transverse spreading, respectively. A decreased degree of transverse spreading on the strips is not compensated for by an increase in longitudinal spreading. Jokingly, one can compare this situation with actors controlling the form of the human body: factors controlling the linear size are quite different from those controlling transverse dimensions.
Possible factors controlling the longitudinal spreading
Factors controlling longitudinal spreading determine the distance between the active edge and central part of fibroblast body. It is likely that the degree of longitudinal spreading is determined by the balance between the action of microtubules stimulating the extension of lamellipods in a longitudinal direction and contractile action of the actin-myosin system in the same direction. This suggestion is supported by the experiments with cytoskeleton-specific drugs, colcemid and cytochalasin D.
Experiments with colcemid show that the microtubular system is essential for maintenance of fibroblast length: this length decreased considerably after depolymerization of microtubules both on the planar substrate and on the strip. These cells were devoid of microtubules and had a collapsed system of intermediate filaments. Their only functioning component of cytoskeleton was the actin-myosin cortex. One may suggest that the mechanism maintaining the length in this situation is based upon the dynamic equilibrium between two activities of this system: the extension of lamellipods based on polymerization of actin microfilaments (Svitkina and Borisy, 1999; Borisy and Svitkina, 2000) and contractile tension of cortex (Cramer, 1999). Possibly, in the course of cell stretching caused by extension and attachment of lamellipods during spreading, centripetal tension increases progressively, until it becomes high enough to stop further elongation.
In contrast to colcemid, cytochalasin D significantly increased the length of fibroblasts both on the planar substrate and on the strips. Cytochalasin D is known to profoundly disorganize the actin system. However, the effects of this drug at the cellular level are far from simple. Spreading of fibroblasts in cytochalasin-containing medium was not completely inhibited during the first 24 hours, but morphology of this spreading is profoundly changed: instead of wide lamellae with microfilament bundles, the cells in this medium formed narrow non-contractile processes packed with microtubules and intermediate filaments and needle-like accumulations of short actin microfilaments at their tips. After 24 hours the growth of these processes stops and they become completely immobile (Bliokh et al., 1980). The ability of cytochalasin-treated cells to extend narrow processes suggests that actin polymerization at their ends is not fully inhibited.
The growth of the processes in cytochalasin-containing medium is almost completely inhibited by the addition of colcemid (Bliokh et al., 1980). Thus, microtubules are essential for promoting their growth. One possible mechanism for this promotion is through the microtubules providing some factors, such as Rac1 protein, that enhance actin polymerization and lamellipod formation (Waterman-Storer et al., 1999).
In the control fibroblast with an intact microtubular and actin-myosin cytoskeleton, these two systems interact to establish the characteristic length of the cell; this length was found to be intermediate between that of cytochalasin-treated cells and colcemid-treated cells. On the basis of the data discussed above, we suggest that this interaction includes competition between two groups of cytoskeletal mechanisms the centrifugal growth of microtubules promoting actin polymerization and extension of lamellipodia and the centripetally directed contractile tension of the actin-myosin system, which may counteract and eventually balance the centrifugal growth. Manifestations of the competition between microtubular growth and actin-myosin contractility are well known (Waterman-Storer and Salmon, 1999). One particularly well known manifestation of this competition was demonstrated in the experiments showing that actin-myosin contractility is increased by drug-induced depolymerization of microtubules (Danowski, 1989; Pletjushkina et al., 1998; Liu et al., 1998; Elbaum et al., 1999).
Possible factors controlling transverse spreading of fibroblasts
In contrast to longitudinal spreading (which determines cell length), transverse spreading determines the width of the active edge. The following hypothetical model of transverse spreading can be proposed. Attachment of each lamellipod at the active edge is followed by the formation of new lamellipods. These lamellipods are extended not only radially, that is, along the direction of longitudinal spreading, but also in perpendicular tangential directions. Attachment of these tangential lamellipods leads to gradual widening of the active cell edge. At least two factors are essential for this component of spreading. One obvious factor is the availability of sufficient area of adhesive substrate surface for attachment of transverse lamellipods. Another factor is the transversely directed contractility of the actin-myosin cortex. This contractility, caused by tangentially oriented actin microfilaments in the lamella, may limit the width of this lamella and of the active edge. Lower the transverse contractility, wider the edge. This contractility may be much higher in the central parts of the fibroblast body than in the leading lamella owing to the higher concentrations of myosin II (Verhovsky et al., 1999). Microtubules may increase transverse contractility, especially, in the central parts of the cells body; for instance, as suggested by Waterman-Storer and Salmon microtubules in this part may release some Rho-activating factor activating myosin contractility (Waterman-Storer and Salmon, 1999). These differences in transverse contractility may be responsible for the fan-like shape of fibroblasts.
Epitheliocytes have no mechanism for the control of cell length
Experiments with three lines of epitheliocytes show that mechanisms controlling the shape of single discoid cells of this tissue type are significantly different from those of fibroblasts. When squeezed on the linear strip epitheliocytes acquired an ellipsoid shape with a maximal length considerably higher than the diameter of discoid cells on the planar substrate. In the experiments with MDCK cells, these changes to the length proved to be reversible: these cells were restored to their original epithelial shape 24 hours after return from the strips to the planar substrate. Colcemid had no significant effects on the cell length on the strip. The shape of epitheliocytes on the strips remained smoothly elliptical but they did not form stable lateral edges or narrow waists proximal to lamellae.
Thus, as expected, epitheliocytes did not undergo microtubule-dependent polarization even in the conditions where substrate shape maximally favoured elongation.
Obviously epitheliocytes have no control over their maximal length. During their spreading, longitudinal and transverse directions are not distinguished. Most probably, these cells form and attach lamellipods in all possible directions eventually acquiring a discoid or ellipsoid shape. It is well known that spatial patterns of microfilament bundles and of the microtubular system are quite different in epitheliocytes and in fibroblasts. These differences extend to microtubular dynamics in cytoplasts prepared from the cells of these two types (Rodionov et al., 1999). Probably, differences in control of cell length between epitheliocytes and fibroblasts are due to these differences in cytoskeletal organization. However, the exact factors responsible for these differences are not known.
One cannot also exclude that, beside balanced dynamic interaction of cytoskeletal structures, there are other factors controlling cell length in fibroblasts and these may be absent in epitheliocytes. For instance, anchorage modulations of protein synthesis on the planar substrate and on the strip may be involved in this control.
To summarize, the experiments presented in this paper show the existence of cell-length control and cell-specific differences in this control. Cultivation of cells on narrow linear strips of adhesive substrate may provide a convenient experimental system for furthering the analysis of these phenomena.
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ACKNOWLEDGMENTS |
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