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Effect on the adhesion and locomotion of mouse fibroblasts by their interacting with differently charged substrates
Copyright 0 1981 by Academic Press. Inc. All rights of reproductmn in any form reserved 0014.4827/811090039-07$02.(K)/O
EFFECT
ON THE ADHESION FIBROBLASTS DIFFERENTLY
AND
BY THEIR
LOCOMOTION
INTERACTING
CHARGED
YUKITAKA
OF MOUSE WITH
SUBSTRATES
SUGIMOTO
SUMMARY Attachment of fibroblastic cells to differently charged substrates was observed by electron microscopy. Cells attached with large contact regions to less negatively charged substrates. When the negativity of the substrate charge increased. the total area of the contact regions decreased. The degree of adhesion was estimated by the ‘contact index’. which is the percentage of the contact regions in comparison with the circumference of the cells in vertical sections. The results in this paper show that the contact index has a fine correlation with both the substrate charge and cell locomotion. This indicates that electrostatic interaction at the cell surface is an important factor in controlling cell locomotion and cell adhesion.
The density and distribution of negatively charged sites on a cell surface membrane are implicated in cell contact phenomenacell recognition, contact and subsequent interaction [l-3]. In the early morphogenesis of an amphibian embryo, morphogenetically active cells undergo a rapid increase in surface charge with the onset of gastrulation, whereas less active cells retain a constant in surface charge [4]. The metastatic behavior of tumor cells has also been considered to be related to the surface charge [5-81, though some investigators have found no change in surface negativity associated with transformation [7] and active metastatic potentials [8]. Borysenko & Wood [9] have confirmed that the mobility and topographical distribution of the cell surface anions of tumor cells differ from those of normal counterparts. This suggests that the
distribution of charged sites on the cell surface-rather than the overall density-is important in cell contact interaction. However. it is still unclear to what extent surface-charged sites contribute to the control of cell behavior whose underlying mechanism is correlated with surface-mediated properties. In a previous study [IO]. the locomotion rates of mouse fibroblasts, L cells, on differently charged substrates were compared. Cells were immobilized on less negatively charged substrates, but cell locomotion started and became more active with increasing negativity of the substrate charge. This suggests that cell locomotion is greatly affected by electrostatic interaction at the cell contact area. In this paper, an evaluation of the effect of the substrate charge on altering cell adhesion and locomotion is
40
Y. Sugimoto
WI-4
Cl ‘(22
l-4 WI-I l---i c3
c, c,
c6
Fig. /. Schematic presentation of a profile view of a cell attached to a substrate. C,, C,, C,,--- represent the cell surface regions which are at a distance less than 100nm from the substrate.
reported. The degree of adhesion was estimated by a morphological criterion, the ‘contact index’, which provided a quantitative expression of the actual adhesion between a cell and the substrate in situ. Contact indexes were determined using electron micrographs of vertical secions of ceils. MATERIALS
AND METHODS
Substrates Plastic Petri dishes were coated with a mixture of bovine serum albumin (BSA: Armour Pharmaceutical Co.. Chicago, 10%) and glutaraldehyde (0.35%‘). The mixture was allowed to polymerize at ca 20°C for 3 h. This polymer served as a highly negatively charged substrate and was termed ‘BSA substrate’. Some albumin-coated dishes were further treated with polyI.-lysine (Miles-Yeda Ldt., III.; MW 22 700, 0.05 mg/ ml) dissolved in pH 8.4 borate buffer (0. IS M) (‘BSAlys substrate’) or with poly-t-histidine (Miles-Yeda Ldt.; MW II 100, 0.1 mg/ml) dissolved in pH 5.0 borate buffer (0.15 M) (‘BSA-his substrate’) for one day at room temperature. Before use, each substrate was washed with Ca2+- and MgY+-free phosphatebuffered saline (pH 7.2) supplemented with EDTA
Table 1. Comparison
Fix. 2. Measurement of cell length on phase-contrast micrographs. The central point of the major cell region was determined approximately. and six lines were drawn passing through the point, each at an angle of 30”. Those segments were measured.
(PBS-EDTA) for 5 days. The method of preparing these substrates was modified from that of MacieiraCoelho & Avrameas [I I].
Electron microscopy For electron microscopy, 3~ lo” cells in IO ml of Eagle’s minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS) and 0.01 M Hepes adjusted to pH 6.8, 7.0. 7.4 or 8.2 by I N NaOH were seeded into each dish and incubated for I day at 37°C. The culture medium was replaced with 5 ml of fresh Hanks’ solution, and the cultures were placed at 4°C for 20 min. They were then gently rinsed twice in cold Hanks’ solution. Fixation was accomplished in situ with 2.5% glutaraldehyde in a 0.1 M Na-cacodylate buffer adjusted to pH 7.2. After fixation for 30 min at 20°C. the cells were carefully washed with a large volume of Hanks’ solution and post-fixed with I .O% 0~0, in 0.1 M Na-cacodylate buffer (pH 7.2) for 30 min. The cells were then washed in double-distilled water. stained with I c/r aqueous uranyl acetate, dehydrated through a graded series of ethanol and embedded in Epon 812. Using an 8800 ultramicrotome (LKB, Sweden), ultrathin sections perpendicular to the plane of the substrate were made through various parts of the substrate as described by Eguchi & Okada [l2]. Thin
of‘cell len,gth on the electron micrographs
br.ith that on the phmse-
contrast micrographs Cell length (pm) ? S.D. Substrate BSA (pH 7.4) BSA-lys (pH 7.4)
Electron micrograph
Phase-contrast micrograph
20.9k5.91 (n=39)” 28.5+9.96 (n=37)”
21.8k4.86 (n=72)” 31.8fl0.69 (n=72)h
F--test
t-test
O.l
0.4
P>O.5
0. I iPCO.2
’ Number of cell sections with nucleus measured on electron micrographs at a magnification of X7000. ’ Number of segments through a central cell region measured on phase-contrast micrographs at a magnification of X.500. Exp Cd
Res 135 (1981)
I-‘ix. 3. (u) Profile view of L cell attached to the BSAlys substrate: (h) enlargement of the contact region between cell and substrate ((I) x4300; (h) x20000.
sections were mounted on couoer grids and stained with lead acetate according to’ keycolds [ 131. Observations were carried out with an HU 1 ID electron microscope (Hitachi, Tokyo).
To estimate the substrate charge, the surface charge of albumin particles (ca 5 pm in size) ground from masses of BSA polymer was measured by the conventional method of cell electrophoresis. For BSA-lys (or BSA-his) substrate, albumin particles were treated with polylysine (or polyhistidine). Before measurement, all particles were incubated with Hanks’ solution containing 5% FCS at 37°C for 3 h.
Cell locomotion was recorded by time-lapse cinematography. By tracing the paths followed by the nuclei of the cells, the distance travelled by each migrating cell during a period of S-IO h was measured continuously with an opisometer. and the locomotion speeds were calculated.
Conlcrct index The contact index is defined as the percentage of surface regions approaching the substrate within 100 nm (as in fie. I. C,. C,) to the circumferences of the vertical section of the cell. Measurement was made on the electron micrographs at a magnification of 7000. and the contact index was calculated as follows. C.+C,+----------+C,, Contact
index=
circumference section
x 100
of vertical of cell
When the cells are sectioned in a random direction through the central region, the average contact index should be reflected in the area of cell-substrate contact. Thirty or forty cell sections with nucleus were measured in each case. The method for measurine was tested on the BSA substrate (pH 7.4) as a lessadhered condition and on the BSA-lys substrate (pH 7.4) as a well-adhered condition. Fir this purpose. twelve cells were randomly selected from phasecontrast micrographs, and the length of six segments passing through the central point of the cell was measured on each cell (fig. 2). The length of the sections was also measured on the electron micrographs. These E.rp
Cell
Res
135 (1981,
42
Y. Sugimoto ;ip. 4. ((I) Profile view of L cell attached to the BS
cell lengths measured in two ways were shown to be identical statistically (table I). Therefore, the method of measurement is expected to be satisfactory.
RESULTS When L cells were seeded onto the BSAlys substrate, they flattened out and became immobile. Electron micrographs show that they had large regions of close contact, the undersurfaces of which were separated by ca 10 nm from the substrate (fig. 3). However, the undersurfaces of most of the remaining regions were more than 200 nm from the substrate. The contact regions were evenly distributed beneath both the central regon of the cell and the margin, although adhesions of most of the cells cultured on plastic or glass have been Exp Cell Res 135 11981)
shown always to be concentrated near the margin [ 141. Endocytosis or exocytosis were often observed on the undersurface of areas interposing the intimate contacts. The electrophoretic mobility of BSA-lys particles was -0.50~0.01 pm/set/V/cm (kS.E.M. pH 7.4), whereas that of the BSA particles was - 1.13+0.02. These figures mean that the BSA substrate had twice as much negativity as the BSA-lys substrate. On the BSA substrate the cells were nearly spherical in shape, with small thin pseudopodia. Although the contact regions were scarce and very small, the distance between the cell and the substrate at close contact was also ca 10 nm (fig. 4). These cells were actively locomoting at an average speed of 64 pm/h (pH 7.4). On the BSA-
the plastic substrate. The electrophoretic mobilities of BSA-his particles at pH 6.8, 7.4, and 8.2 were -0.62iO.01 ~m/sec/V/ cm (_+S.E.M.), -0.7.5&0.02, and -0.97+ 0.01 respectively; but no marked increase with pH was seen in either the BSA or the BSA-lys particles within the same pH regions. The facts that the surface charge of 2 4 6 810 20 40 the BSA-his substrate depends on pH, but CONTACT INDEX that of the BSA and the BSA-lys substrate Fig. 5. Correlation between contact index and substrate charge and between contact index and cell does not, indicate that those changes in locomotion speeds. contact index are not due to pH itself, but to the substrate charge. In fig. 5. the substrate charge and cell his substrate whose surface charge was intermediate between the BSA substrate locomotion speeds are plotted against the and the BSA-lys substrate, many cells had contact index. Fig. 5 indicates that the conan elongated shape with a few large pseudo- tact indexes have a fine correlation with pods like those seen on plastic substrates, both the substrate charge and the cell locoand were actively locomoting at an average motion. This strongly suggests that electrospeed of 26 pm/h (pH 7.4), which is about static interaction at the cell surface is an the same speed as that of cells on a plastic important factor in controlling the cell’s substrate [lo]. The cell membrane in the locomotion and adhesion. close contact regions was also separated from the substrate by ca 10 nm. DISCUSSION The contact regions were considered to be adhesion sites; thus, the degree of cell- The degree of cell-substrate adhesion has substrate adhesion could be estimated by often been measured by the susceptibility the total area of the contact regions. To of cells to mechanical dislodgement [ISassess the effect of substrate charge on 181.although the force necessary to detach cell adhesion, the degree of adhesion between cell and substrate was evaluated by calculating the contact index from the electron micrographs. As shown in table 2, the highest contact index was found when No. of Contact pH of sections index culture the cells adhered to the BSA-lys substrate, examined Substrate medium (with S.D.) whereas the lowest index was found with the BSA substrate. The contact index of the BSA-lys 6.8 37.6 (9.7) 33 32.4 (7.9) 37 7.4 cells on the BSA-his substrate varied with 33 BSA-his 6.8 37.4 (9.0) the pH, with a high index similar to that 7.0 30.4 (9.6) 36 of the BSA-lys substrate at pH 6.8, ap35 1.4 14.7 (9.3) 36 8.2 5.4 (4.3) proaching that of the BSA substrate when 42 7.4 2.0 (2.3) the pH was raised to 8.2. Changing the pH, BSA 32 14.9 (9.9) 6.X however, had little effect on the contact in- Plastic 17.0 (8.5) 29 7.4 dex of cells on the BSA-lys substrate and
44
Y. Sugimoto
cells from the substrate is not always equivalent to the strength of adhesion [16, 191. In this report, the contact index was designed for estimating the degree of cellsubstrate adhesion. The adhesion sites were observed in separate regions when the cells attached to the substrate [12, 14, 2023]. These sites were characterized by a close (less than 10 nm) approach of the cell plasma membrane to the substrate [ 12, 21241. A region of the cell surface as much as 80 nm away from the substrate may also be an adhesion site, presumably less strong, but more extensive than the area of close approach [25, 261. Thus the total strength of adhesion between cell and substrate can be determined both by the small spots of firm, specialized adhesions and by the weak diffuse adhesion of the relatively large surface area between these spots. The area of contact region in this report, therefore, provides an estimation of adhesion between cell and substrate; the contact index is a relative expression of the degree of cellsubstrate adhesion. The results summarized in fig. 5 show that cell-substrate adhesion is closely related to substrate surface negativity and also suggest that cell locomotion is controlled by cell adhesion. However, so far, the hypothesis that cell adhesion plays an important role in controlling cell locomotion has scarcely been supported by the experimental data. Curtis & Biitiltjens [27] have reported that cell adhesion affects neither the rate of locomotion nor the rate of initiation of pseudopods when the adhesiveness of chick heart fibroblast has been modified by alteration of their lipid composition. However, they had not measured actual cell adhesion. Pouyssegur & Pastan [28] have compared the rate and direction of the motion of an adhesivedeficient mutant and its parental cell. They Erp Cd
RFS 135 (198/J
have shown that the formation of pseudopods and the rate of locomotion do not seem to be altered by a decrease of adhesiveness, whereas the persistence of the direction of locomotion was dramatically reduced. In the condition where cell-substrate adhesion is too weak for the cell to stabilize its locomotory organ at the adhesion sites, the cell must fail to have a normal locomotion on the substrate, even if the cell may move unsteadily owing to the protrusion of pseudopods. In contrast to the above study, this study shows that L cells have an apparently normal locomotion and travel long distances, with occasional changes of direction, even on the BSA substrate where cell adhesion is weakest. The contact index originated for estimating cell-substrate adhesion seems to reflect the degree of adhesion more faithfully than the others often used until now, such as the susceptibility to mechanical dislodgement or to detachment agents or size of cell aggregation. Therefore, the experimental data are reliable, shows fine correlation between degree of cell adhesion and cell locomotion speed. It has recently been reported that actin containing microfilament bundle formation which is inversely correlated with cell locomotion [29-311 is coupled with the formation of intimate adhesion sites [21, 22, 24, 321. This perhaps suggests that the density and distribution of adhesion sites have a more important role in fibroblast locomotion than the strength of the adhesion. The finding that cell locomotion is closely related to the contact index should be explained in that cell locomotion is not directly controlled by the strength of the adhesion, but controlled through a process in which the adhesions influence the formation of microfilament bundles. Grinnell & Hays [33] have reported that polycationic compounds as well as other
ligands promote cell spreading by causing an interaction between the cell surface and the ligands adsorbed onto a substrate and suggested that the ligand-promoted spreading is a general cellular response to specific cell-substrate interactions rather than a result of binding between a unique cell surface receptor and the substrate. Therefore, the spreading of the cells too flatly, with the resulting immobilization on the BSA-lys substrate, is considered to be due to a general cellular response. The results in this report indicate that the electrostatic interaction at the cell surface is sufficient to induce a considerable change in spreading and locomoting behavior. This suggests that the surface-charged sites which have a specific distribution on the cell surface could control the cell behavior in a specific manner by interacting with other cells in vivo. Culture cells generally change from a spread to a round shape when cell division occurs. The very flat-spread cells on the BSA-lys substrate also rounded up during the period of cell division [IO]. However, this drastic morphological change indicates that some reorganization of the surface components including charged sites, if any, may perhaps be associated with division. Evidence in favor of this possibility is provided by the fact that a particular antiserum is cytotoxic to spread cells but not to round cells [33. 34). The author is very grateful to Dr G. Eguchi for guiding the electron microscopy with great skill and enthusiasm and for critical reading of the manuscript.
6. 7. 8. 9. IO. I I. 12. 13. 14. IS. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26. 27.
2X. 29. 30. 31. 32. 33. 34.
REFERENCES I. Weiss, L 2. Weiss, L 3. Grinnell, J ceil biol
Printed
in Sweden
& Ziegel. R, J theor biol 34 ( 1972) ?I. & Subjeck, J R, J cell sci I4 (1974) 215. F, Tobelman, M Q & Hackenbrock, C R, 66 (I 975) 470.
Schaeffer, B E, Schaeffer, H E & Brick, I, Dev biol 34 (1973) 66. Ambrose, E J. James. A M & Lowick, J H. Nature 177 (1956) 576. Forrester. J A. Ambrose. E J & Stocker, M G, Nature 201 (1964) 945. Latner. A L 61 Turner. G A. .I cell xci 14 (1974) 203. Raz. A. Bucana, C, McLellan, W & Fidler, I J, Nature 284 (1980) 363. Borvsenko. J Z & Wood, W. EXP cell res I I8 (lY?Y) 215. Suaimoto. Y & Haeiwara. A. Exn cell res 120 (1527% 245. Macieira-Coelho. A & Avrameas. S. Proc natl acad sci US 69 (1972) 2469. Eguchi, G & Okada. T S. Dev growth differ I2 (1971) 297. Reynolds, E S. J cell biol I7 (1963) 20X. Harris, A. Dev biol 35 (1973) 97. Coman. D R, Cancer res 4 (1944) 625. Weiss. L, Theoret biol 2 (1962) 236. Gail. M H & Boone, Ch W. Exp cell res 70 ( 1972) 33. Couchman. J R & Rees, I> .4. Cell biol int rep 3 (1979) 431. Steinberg, M S. Cellular membranes in development (ed M Locke) pp. 321-366. Academic Press, New York (1964). Revel. J P & Wolken, K. Exp cell res 7X (1973) 1. Abercrombie. M, Heaysman. J E M & Pegrum. S M, Exp ceil res 67 (1971) 3SY. Heath, J P & Dunn, G A, J cell sci 29 (1978) 197. Cornell, R. Exp cell res 58 (1969) 289. Rees. D A, Badley, R A, Lloyd, C W. Thorn. D & Smith. C G. Sot exo biol symu 32 (1978) 241. Lochner. L & Iz&rd. C s, j cell biol 59 (1973) abstr. 199a. Abercrombie. M & Dunn, G A. Exp cell res 92 (1975) 57. Curtis. A S G & Biiiiltjens. T E J, Locomotion of tissue cells, Ciba foundation symposium 14, pp. 171-179. Elsevier. Excerpta Medica. and NorthHolland, Amsterdam (1973). Pouyssegur. .I & Pastan, I. Exp cell re\ 121 (IY~YJ 373. Wehland, J, Osborn. M & Weber. K. Proc natl acad sci US 74 (1977) 5613. Wehland. J. Stockem. W 8: Weber. K. Exn cell reb 115(1978)451. Couchman. J R & Rees. D A. J cell sci 39 (1979) 149. Branina. E E. Vasiliev. Ju M & Gelfand. I M. Exn cellyes 97 (1976) 341. Grinnell, F & Hays. D G. Exp cell res I I6 (197X) ,-I< L ,A. Ishii. K. Mizumoto, T, Hanaoka, M sumoto. A. Exp cell res 105 (1977) 237.
Received December 3 I, 1980 Revised version received March Accepted March 31. 1981
24, 1981
&
Mat-
EFFECT
ON THE ADHESION FIBROBLASTS DIFFERENTLY
AND
BY THEIR
LOCOMOTION
INTERACTING
CHARGED
YUKITAKA
OF MOUSE WITH
SUBSTRATES
SUGIMOTO
SUMMARY Attachment of fibroblastic cells to differently charged substrates was observed by electron microscopy. Cells attached with large contact regions to less negatively charged substrates. When the negativity of the substrate charge increased. the total area of the contact regions decreased. The degree of adhesion was estimated by the ‘contact index’. which is the percentage of the contact regions in comparison with the circumference of the cells in vertical sections. The results in this paper show that the contact index has a fine correlation with both the substrate charge and cell locomotion. This indicates that electrostatic interaction at the cell surface is an important factor in controlling cell locomotion and cell adhesion.
The density and distribution of negatively charged sites on a cell surface membrane are implicated in cell contact phenomenacell recognition, contact and subsequent interaction [l-3]. In the early morphogenesis of an amphibian embryo, morphogenetically active cells undergo a rapid increase in surface charge with the onset of gastrulation, whereas less active cells retain a constant in surface charge [4]. The metastatic behavior of tumor cells has also been considered to be related to the surface charge [5-81, though some investigators have found no change in surface negativity associated with transformation [7] and active metastatic potentials [8]. Borysenko & Wood [9] have confirmed that the mobility and topographical distribution of the cell surface anions of tumor cells differ from those of normal counterparts. This suggests that the
distribution of charged sites on the cell surface-rather than the overall density-is important in cell contact interaction. However. it is still unclear to what extent surface-charged sites contribute to the control of cell behavior whose underlying mechanism is correlated with surface-mediated properties. In a previous study [IO]. the locomotion rates of mouse fibroblasts, L cells, on differently charged substrates were compared. Cells were immobilized on less negatively charged substrates, but cell locomotion started and became more active with increasing negativity of the substrate charge. This suggests that cell locomotion is greatly affected by electrostatic interaction at the cell contact area. In this paper, an evaluation of the effect of the substrate charge on altering cell adhesion and locomotion is
40
Y. Sugimoto
WI-4
Cl ‘(22
l-4 WI-I l---i c3
c, c,
c6
Fig. /. Schematic presentation of a profile view of a cell attached to a substrate. C,, C,, C,,--- represent the cell surface regions which are at a distance less than 100nm from the substrate.
reported. The degree of adhesion was estimated by a morphological criterion, the ‘contact index’, which provided a quantitative expression of the actual adhesion between a cell and the substrate in situ. Contact indexes were determined using electron micrographs of vertical secions of ceils. MATERIALS
AND METHODS
Substrates Plastic Petri dishes were coated with a mixture of bovine serum albumin (BSA: Armour Pharmaceutical Co.. Chicago, 10%) and glutaraldehyde (0.35%‘). The mixture was allowed to polymerize at ca 20°C for 3 h. This polymer served as a highly negatively charged substrate and was termed ‘BSA substrate’. Some albumin-coated dishes were further treated with polyI.-lysine (Miles-Yeda Ldt., III.; MW 22 700, 0.05 mg/ ml) dissolved in pH 8.4 borate buffer (0. IS M) (‘BSAlys substrate’) or with poly-t-histidine (Miles-Yeda Ldt.; MW II 100, 0.1 mg/ml) dissolved in pH 5.0 borate buffer (0.15 M) (‘BSA-his substrate’) for one day at room temperature. Before use, each substrate was washed with Ca2+- and MgY+-free phosphatebuffered saline (pH 7.2) supplemented with EDTA
Table 1. Comparison
Fix. 2. Measurement of cell length on phase-contrast micrographs. The central point of the major cell region was determined approximately. and six lines were drawn passing through the point, each at an angle of 30”. Those segments were measured.
(PBS-EDTA) for 5 days. The method of preparing these substrates was modified from that of MacieiraCoelho & Avrameas [I I].
Electron microscopy For electron microscopy, 3~ lo” cells in IO ml of Eagle’s minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS) and 0.01 M Hepes adjusted to pH 6.8, 7.0. 7.4 or 8.2 by I N NaOH were seeded into each dish and incubated for I day at 37°C. The culture medium was replaced with 5 ml of fresh Hanks’ solution, and the cultures were placed at 4°C for 20 min. They were then gently rinsed twice in cold Hanks’ solution. Fixation was accomplished in situ with 2.5% glutaraldehyde in a 0.1 M Na-cacodylate buffer adjusted to pH 7.2. After fixation for 30 min at 20°C. the cells were carefully washed with a large volume of Hanks’ solution and post-fixed with I .O% 0~0, in 0.1 M Na-cacodylate buffer (pH 7.2) for 30 min. The cells were then washed in double-distilled water. stained with I c/r aqueous uranyl acetate, dehydrated through a graded series of ethanol and embedded in Epon 812. Using an 8800 ultramicrotome (LKB, Sweden), ultrathin sections perpendicular to the plane of the substrate were made through various parts of the substrate as described by Eguchi & Okada [l2]. Thin
of‘cell len,gth on the electron micrographs
br.ith that on the phmse-
contrast micrographs Cell length (pm) ? S.D. Substrate BSA (pH 7.4) BSA-lys (pH 7.4)
Electron micrograph
Phase-contrast micrograph
20.9k5.91 (n=39)” 28.5+9.96 (n=37)”
21.8k4.86 (n=72)” 31.8fl0.69 (n=72)h
F--test
t-test
O.l
0.4
P>O.5
0. I iPCO.2
’ Number of cell sections with nucleus measured on electron micrographs at a magnification of X7000. ’ Number of segments through a central cell region measured on phase-contrast micrographs at a magnification of X.500. Exp Cd
Res 135 (1981)
I-‘ix. 3. (u) Profile view of L cell attached to the BSAlys substrate: (h) enlargement of the contact region between cell and substrate ((I) x4300; (h) x20000.
sections were mounted on couoer grids and stained with lead acetate according to’ keycolds [ 131. Observations were carried out with an HU 1 ID electron microscope (Hitachi, Tokyo).
To estimate the substrate charge, the surface charge of albumin particles (ca 5 pm in size) ground from masses of BSA polymer was measured by the conventional method of cell electrophoresis. For BSA-lys (or BSA-his) substrate, albumin particles were treated with polylysine (or polyhistidine). Before measurement, all particles were incubated with Hanks’ solution containing 5% FCS at 37°C for 3 h.
Cell locomotion was recorded by time-lapse cinematography. By tracing the paths followed by the nuclei of the cells, the distance travelled by each migrating cell during a period of S-IO h was measured continuously with an opisometer. and the locomotion speeds were calculated.
Conlcrct index The contact index is defined as the percentage of surface regions approaching the substrate within 100 nm (as in fie. I. C,. C,) to the circumferences of the vertical section of the cell. Measurement was made on the electron micrographs at a magnification of 7000. and the contact index was calculated as follows. C.+C,+----------+C,, Contact
index=
circumference section
x 100
of vertical of cell
When the cells are sectioned in a random direction through the central region, the average contact index should be reflected in the area of cell-substrate contact. Thirty or forty cell sections with nucleus were measured in each case. The method for measurine was tested on the BSA substrate (pH 7.4) as a lessadhered condition and on the BSA-lys substrate (pH 7.4) as a well-adhered condition. Fir this purpose. twelve cells were randomly selected from phasecontrast micrographs, and the length of six segments passing through the central point of the cell was measured on each cell (fig. 2). The length of the sections was also measured on the electron micrographs. These E.rp
Cell
Res
135 (1981,
42
Y. Sugimoto ;ip. 4. ((I) Profile view of L cell attached to the BS
cell lengths measured in two ways were shown to be identical statistically (table I). Therefore, the method of measurement is expected to be satisfactory.
RESULTS When L cells were seeded onto the BSAlys substrate, they flattened out and became immobile. Electron micrographs show that they had large regions of close contact, the undersurfaces of which were separated by ca 10 nm from the substrate (fig. 3). However, the undersurfaces of most of the remaining regions were more than 200 nm from the substrate. The contact regions were evenly distributed beneath both the central regon of the cell and the margin, although adhesions of most of the cells cultured on plastic or glass have been Exp Cell Res 135 11981)
shown always to be concentrated near the margin [ 141. Endocytosis or exocytosis were often observed on the undersurface of areas interposing the intimate contacts. The electrophoretic mobility of BSA-lys particles was -0.50~0.01 pm/set/V/cm (kS.E.M. pH 7.4), whereas that of the BSA particles was - 1.13+0.02. These figures mean that the BSA substrate had twice as much negativity as the BSA-lys substrate. On the BSA substrate the cells were nearly spherical in shape, with small thin pseudopodia. Although the contact regions were scarce and very small, the distance between the cell and the substrate at close contact was also ca 10 nm (fig. 4). These cells were actively locomoting at an average speed of 64 pm/h (pH 7.4). On the BSA-
the plastic substrate. The electrophoretic mobilities of BSA-his particles at pH 6.8, 7.4, and 8.2 were -0.62iO.01 ~m/sec/V/ cm (_+S.E.M.), -0.7.5&0.02, and -0.97+ 0.01 respectively; but no marked increase with pH was seen in either the BSA or the BSA-lys particles within the same pH regions. The facts that the surface charge of 2 4 6 810 20 40 the BSA-his substrate depends on pH, but CONTACT INDEX that of the BSA and the BSA-lys substrate Fig. 5. Correlation between contact index and substrate charge and between contact index and cell does not, indicate that those changes in locomotion speeds. contact index are not due to pH itself, but to the substrate charge. In fig. 5. the substrate charge and cell his substrate whose surface charge was intermediate between the BSA substrate locomotion speeds are plotted against the and the BSA-lys substrate, many cells had contact index. Fig. 5 indicates that the conan elongated shape with a few large pseudo- tact indexes have a fine correlation with pods like those seen on plastic substrates, both the substrate charge and the cell locoand were actively locomoting at an average motion. This strongly suggests that electrospeed of 26 pm/h (pH 7.4), which is about static interaction at the cell surface is an the same speed as that of cells on a plastic important factor in controlling the cell’s substrate [lo]. The cell membrane in the locomotion and adhesion. close contact regions was also separated from the substrate by ca 10 nm. DISCUSSION The contact regions were considered to be adhesion sites; thus, the degree of cell- The degree of cell-substrate adhesion has substrate adhesion could be estimated by often been measured by the susceptibility the total area of the contact regions. To of cells to mechanical dislodgement [ISassess the effect of substrate charge on 181.although the force necessary to detach cell adhesion, the degree of adhesion between cell and substrate was evaluated by calculating the contact index from the electron micrographs. As shown in table 2, the highest contact index was found when No. of Contact pH of sections index culture the cells adhered to the BSA-lys substrate, examined Substrate medium (with S.D.) whereas the lowest index was found with the BSA substrate. The contact index of the BSA-lys 6.8 37.6 (9.7) 33 32.4 (7.9) 37 7.4 cells on the BSA-his substrate varied with 33 BSA-his 6.8 37.4 (9.0) the pH, with a high index similar to that 7.0 30.4 (9.6) 36 of the BSA-lys substrate at pH 6.8, ap35 1.4 14.7 (9.3) 36 8.2 5.4 (4.3) proaching that of the BSA substrate when 42 7.4 2.0 (2.3) the pH was raised to 8.2. Changing the pH, BSA 32 14.9 (9.9) 6.X however, had little effect on the contact in- Plastic 17.0 (8.5) 29 7.4 dex of cells on the BSA-lys substrate and
44
Y. Sugimoto
cells from the substrate is not always equivalent to the strength of adhesion [16, 191. In this report, the contact index was designed for estimating the degree of cellsubstrate adhesion. The adhesion sites were observed in separate regions when the cells attached to the substrate [12, 14, 2023]. These sites were characterized by a close (less than 10 nm) approach of the cell plasma membrane to the substrate [ 12, 21241. A region of the cell surface as much as 80 nm away from the substrate may also be an adhesion site, presumably less strong, but more extensive than the area of close approach [25, 261. Thus the total strength of adhesion between cell and substrate can be determined both by the small spots of firm, specialized adhesions and by the weak diffuse adhesion of the relatively large surface area between these spots. The area of contact region in this report, therefore, provides an estimation of adhesion between cell and substrate; the contact index is a relative expression of the degree of cellsubstrate adhesion. The results summarized in fig. 5 show that cell-substrate adhesion is closely related to substrate surface negativity and also suggest that cell locomotion is controlled by cell adhesion. However, so far, the hypothesis that cell adhesion plays an important role in controlling cell locomotion has scarcely been supported by the experimental data. Curtis & Biitiltjens [27] have reported that cell adhesion affects neither the rate of locomotion nor the rate of initiation of pseudopods when the adhesiveness of chick heart fibroblast has been modified by alteration of their lipid composition. However, they had not measured actual cell adhesion. Pouyssegur & Pastan [28] have compared the rate and direction of the motion of an adhesivedeficient mutant and its parental cell. They Erp Cd
RFS 135 (198/J
have shown that the formation of pseudopods and the rate of locomotion do not seem to be altered by a decrease of adhesiveness, whereas the persistence of the direction of locomotion was dramatically reduced. In the condition where cell-substrate adhesion is too weak for the cell to stabilize its locomotory organ at the adhesion sites, the cell must fail to have a normal locomotion on the substrate, even if the cell may move unsteadily owing to the protrusion of pseudopods. In contrast to the above study, this study shows that L cells have an apparently normal locomotion and travel long distances, with occasional changes of direction, even on the BSA substrate where cell adhesion is weakest. The contact index originated for estimating cell-substrate adhesion seems to reflect the degree of adhesion more faithfully than the others often used until now, such as the susceptibility to mechanical dislodgement or to detachment agents or size of cell aggregation. Therefore, the experimental data are reliable, shows fine correlation between degree of cell adhesion and cell locomotion speed. It has recently been reported that actin containing microfilament bundle formation which is inversely correlated with cell locomotion [29-311 is coupled with the formation of intimate adhesion sites [21, 22, 24, 321. This perhaps suggests that the density and distribution of adhesion sites have a more important role in fibroblast locomotion than the strength of the adhesion. The finding that cell locomotion is closely related to the contact index should be explained in that cell locomotion is not directly controlled by the strength of the adhesion, but controlled through a process in which the adhesions influence the formation of microfilament bundles. Grinnell & Hays [33] have reported that polycationic compounds as well as other
ligands promote cell spreading by causing an interaction between the cell surface and the ligands adsorbed onto a substrate and suggested that the ligand-promoted spreading is a general cellular response to specific cell-substrate interactions rather than a result of binding between a unique cell surface receptor and the substrate. Therefore, the spreading of the cells too flatly, with the resulting immobilization on the BSA-lys substrate, is considered to be due to a general cellular response. The results in this report indicate that the electrostatic interaction at the cell surface is sufficient to induce a considerable change in spreading and locomoting behavior. This suggests that the surface-charged sites which have a specific distribution on the cell surface could control the cell behavior in a specific manner by interacting with other cells in vivo. Culture cells generally change from a spread to a round shape when cell division occurs. The very flat-spread cells on the BSA-lys substrate also rounded up during the period of cell division [IO]. However, this drastic morphological change indicates that some reorganization of the surface components including charged sites, if any, may perhaps be associated with division. Evidence in favor of this possibility is provided by the fact that a particular antiserum is cytotoxic to spread cells but not to round cells [33. 34). The author is very grateful to Dr G. Eguchi for guiding the electron microscopy with great skill and enthusiasm and for critical reading of the manuscript.
6. 7. 8. 9. IO. I I. 12. 13. 14. IS. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26. 27.
2X. 29. 30. 31. 32. 33. 34.
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Received December 3 I, 1980 Revised version received March Accepted March 31. 1981
24, 1981
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