Cell locomotion on differently charged substrates

Printed m Sweden CopyrIght 0 1979 by Academic Pree. Inc. All rights of reproduction in any form reerved oOl4-48?7/79/o6n?45-OX$O~.~/O

Experimental

CELL

Cell Research 120 (1979) 245-252

LOCOMOTION ON DIFFERENTLY CHARGED SUBSTRATES

Effects of Substrate Charge on Locomotive Speed of Fibroblastic Cells YUKITAKA Laboratory

SUGIMOTO and ATSUYOSHI

HAGIWARA’

of Developmental Biology, Zoological Institute, Faculty of Science, University of Kyoto, Sakyo-ku, Kyoto 606, Japan

SUMMARY To assess how cell locomotive behavior is influenced by an electrostatic interaction at the cells’ contact area, locomotion speeds of mouse tibroblast, L cells, were compared on differently charged non-living substrates. These substrates were prepared by polymerizing bovine serum albumin with glutaraldehyde, and their surface charge was changed by treating them with poly-Llysine or poly-L-histidine. The locomotion speeds increased with increasing negativity of the substrate charge. On the less negatively charged substrates, the cells ceased locomotion and did not alter their positions. In spite of the diversity in the cell behavior, there was little difference in cell growth among the different substrates. When the substrates were treated with trinitrobenzene sulphonic acid (TNBS), which reacts with amino and other cationic groups, the immobilization no longer occurred. This indicates that the substrate charge is a main factor in modulating cell behavior.

Many features of cell behavior are contact interaction phenomena since they take place only when the cell is in contact with a substrate or meets a new substrate such as another cell [l]. Studies on cell locomotion and adhesion of fibroblasts have revealed that motile behaviors such as direction and speed are affected by cell-substrate adhesion [2, 31, that is, contact interaction. Cell surface charge has been implicated in the regulation of many different types of contact interaction [4]. In fact, measurements of cell surface charge have in some cases revealed a correlation between changes in net surface charge and altered states of cellular adhesion [5-8]. Thus, it is significant to study how cell behavior is influenced when the cells are subjected to an

electrostatic interaction at the cells’ contact area with non-living substrates or with other cell surfaces. In this study, we have observed cell behavior or variously charged substrates and assessed the effect on cell locomotion of altering the electric charge of the substrate. For these experiments, an albumin polymer was prepared by modifying the method of Macieira-Coelho & Avrameas [9]. The surface charge of this polymer was varied by treating it with homopolymer of amino acids with different isoelectric points. The net surface charge to which cells were actually reacting was estimated by electrophoretic mobilities of the particles of the ’ Deceased: 27 August, 1976. Exp Cell Res IN (IY~YJ

246

Sugimoto and Hagiwara

Table 1. Electrophoretic mobility as a function ofpH for BSA-lys, BSA-his, and BSA particles with.fresh mouse erythrocytes as a reference pH of suspending medium

Particles Preincubated BSA-lys

in Hanks’ solution 6.6 7.4 8.2

Mean mobility ~m/sec/V/cm IL S.E.M. (No. of obj.) +5 % FCS -0.45+0.02 -OSO+O.Ol -0.55+0.01

(67) } (85) (71))

0.1 >P>O.OS

P
BSA-his

6.6 1.4 8.2

-0.60+0.01 -0.75+0.02 -0.97*0.01

(99) } (93) (95) }

BSA

6.6 7.4 8.2

-1.13+0.01 -1.13+0.02 -l.l8ri-0.01

(116)) (116) (104) }

7.4 7.4

+ I .38+0.02 - I .46+0.01

Non-preincubated BSA-lys BSA

Fresh mouse erythrocytes 6.6 7.4 8.2

t-test

-1.11+0.01 - I. I I +0.01 -I .06+0.01

0. I >P>O.O5

PP>O.8 0. I>P>O.O5

(86) 102)

(8% (88) -

(91)

albumin polymer. Locomotive speeds were measured by time-lapse cinematography. To make sure that the cells rext to the charge of the substrate, the effect 01’ the positively charged amino sites of polylysine-treated albumin substrate on cell locomotion was determined by trinitrobenzene sulphonic acid (TNBS)-treatment.

dish was washed for I day with 5 ml of 0.15 M borate buffer at pH 8.2. If necessary, the polymer was then treated with polycation by the procedure given in the following sections. The albumin polymer was washed for 8 days with 5 ml of Ca’+- and Mg*+-free phosphate buffered saline (pH 7.2) supplemented with EDTA (PBS-EDTA). The buffer was changed every day. Before the polymer was seeded with cells, it was washed twice with the assay medium. This polymer was termed the BSA substrate.

Lysine treatment of the albumin polymer MATERIALS

AND METHODS

Cell culture L cells, mouse fibroblasts, were grown in Eagle’s minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS) and were routinely maintained in monolayer on borosilicate glass.

Before the wash with PBS-EDTA, the albumin polymer was incubated with 5 ml of poly-L-lysine HBr (Miles-Yeda Let, Illinois, mol. wt 22700) (0.1 mg/ml) dissolved in borate buffer (pH 8.4) for I day at 20°C. The washing with PBS-EDTA was then continued as above. This polymer was termed the BSA-lys substrate.

Preparation of the albumin polymer

Histidine treatment of the albumin polymer

Equal portions of an aqueous solution of 20% bovine serum albumin (Armour Pharmaceutical Co., Chicago) and an aqueous solution of 0.7 % glutaraldehyde were mixed with constant stirring at 4°C. An aliquot (0.3 ml) of the mixture was poured onto a 6 cm plastic dish (Falcon Plastics, Los Angeles) and spread on the surface with an L-shaped glass rod. After 3 h of polymeiization at room temperature (ca 2O”C), the coated

After polymerization, a low pH borate buffer (0.15 M, pH 5.0) was used instead of the buffer at pH 8.4. After I day, the albumin polymer was incubated with 5 ml of poly-L-histidine (Sigma Chemical Co., St Louis, mol. wt 10000) (0.25 mg/ml) dissolved in the low pH borate buffer for 1 day at 20°C. The washing was then continued as in the procedure above. This polymer was termed the BSA-his substrate.

Substrate charge and cell locomotion

247

3. L cells incubated for ca 24 h on the BSA-lys substrate. Most of the cells have ceased locomotion. The rounded cells are in the mitotic phase. Fig. 4. L cells incubated for ca 24 h on the plastic substrate. Most cells are locomoting actively.

Fig.

I. L cells incubated for 3 h on the BSA substrate. All cells are floating and many have collided with each other to form aggregates. Fig. 2. L cells incubated for ca 24 h on the BSA substrate. The cells have attached to the substrate and are locomoting actively in random directions by means of short-lived pseudopods.

Fig.

Particle electrophoresis

population, which perhaps was in the mitotic phase, was used in this experiment. The cells were collected from unconfluent culture by seesawing at approx. 40 returns per min at 37°C for I h. They were then washed twice with Eagle’s MEM supplemented with S’Z FCS and 0.01 M HEPES adjusted to appropriate pH by I N NaOH (“assay medium”). With IO ml of the assay medium, 2-3~ IO5 cells were seeded into each of the variously coated dishes and incubated at 37°C. Cell locomotion was recorded on 16 mm tine film with 2 min intervals between frames. By tracing the paths followed by nuclei of the cells, the distance travelled by each migrating cell during a period of 5-10 h was measured continuously by an opisometer, and the locomotion speeds were calculated.

Masses of BSA polymer were ground by a homogenizer in the borate buffer (pH 8.2) into particles having a size ca 5 pm. The BSA particles were treated with polylysine or polyhistidine as described above and washed with PBS-EDTA. The non-treated and the polycation-treated BSA particles were preincubated in Hanks’ solution +S% FCS for 3 h at 37”C, then washed and suspended in Hanks’ solution. These Hanks’ solutions were buffered to appropriate pH with 0.01 M HEPES. The electrophoretic mobilities were measured in a rectangular and horizontal cell apparatus of the type described by Oshima [IO]. All measurements were made in serum-free Hanks’ solution at 32°C. with reversal of current, in a known voltage gradient.

Measurement of cell locomotion

Serum pretreatment and TNBS treatment of substrates

To avoid a disturbance of the cell surface that would have resulted if the cells were treated with a proteolytic enzyme [ 11, 121, only the non-attached cell

BSA polymer coated and non-coated plastic dishes were preincubated with MEM with and without 5% FCS for 24 h at 37°C. The dishes were washed twice

248

Sugimoto and Hagiwara maintained the highest surface negative charge among the three kinds of particles, while BSA-lys particles maintained the lowest. The surface charge of both BSA and BSA-lys particles as a function of pH values changed very little. The surface negativity of polyhistidine-treated BSA (BSA-his) particles increased within the range between the above two with increasing pH values. This increase was highly significant statistically.

105

105:

Fig. 5. Abscissa: time (days); ordinate: cell density/ 6 cm Petri dish. Growth of L cells on the BSA substrate (- - -), on the BSA-Iys substrate (---) and on the plastic substrate (- -- -). The cells with 5 ml of assay medium buffered at pH 7.2 were seeded into each dish and incubated at 37°C. The cells were harvested from each dish every day by trypsinizing and were counted using a FucksRosental hemocytometer.

with phosphate-buffered saline (PBS) at pH 8.0 and were incubated for I h at 20°C with or without 5 ml of trinitrobenzene sulphonic acid (TNBS) (50 pg/ml) dissolved in PBS. After this treatment, they were washed three times with PBS.

RESULTS Particle electrophoresis of albumin polymer As shown in table 1, the anodal mobility of BSA particles was considerably reduced following incubation with serum-containing Hanks’ solution. In the case of polylysinetreated BSA (BSA-lys) particles, serum treatment reversed the direction of electrophoretic migration from cathodal to anodal. In spite of this change, which may be due to adsorption of serum constituents, the mobility of the BSA-lys particles was less than 47% of that of the BSA particles even after preincubation with serum-containing solution. When the pH of both the preincubating and suspending solutions was changed in the pH range 6.6-8.2, BSA particles

Modulation of cell behavior by the differently charged substrates When L cells with the assay medium adjusted to pH 7.2 were seeded onto the BSA substrate, the cells did not adhere to the substrate for the first l&20 h of incubation, but collided with each other to form aggregates (fig. 1). Subsequently, the single cells and aggregates started to adhere to the BSA substrate, and the aggregates dis-

Table 2. Speed of cell locomotion on BSAhis substrates as a function of pH changes

PH

Average speed (pm/h)

S.D.

No. of cells measured

Average duration of measuring for each cell (hours)

38 21 20 I2 I7 II 16

9.6 9.7 9.9 8.4 6.1 8.7 6.8

28 34 22 20 18 II 13

10.0 10.0 9.3 9.5 7.7 6.9 7.9

First day of incubution 6.6 6.8 7.0 7.2 7.4 7.8 8.2

5.3 6.8 6.1 25.5 32.8 33.1 47.4

3.2 5.3 5.0 8.3 14.4 7.0 20.0

Second day of incubation 6.6 6.8 7.0 7.2 7.4 7.8 8.2

0.7 2.4 6.2 9.1 25.9 33.9 41.3

0.7 2.2 4.0 5.9 6.5 10.3 11.3

Substrate

Table 3. Speed of cell locomotion on the BSA substrates as a function ofpH changes Average speed (pm/h)

No. of cells measwed

Average duration of measuring for each cell (hours)

charge and cell locomotion

249

ponent(s) are therefore required for cell locomotion as reported in previous studies [ 13-161.

Effects of the substrate charge on cell locomotion S.D. PH On the BSA-his substrate, the cells adhered Srcond day of incubation and spread at a pH lower than 7.4 and 6.5 6.6 57.0 16.2 19 rounded up above pH 7.8. The cells ceased 25 5.1 7.0 60.4 21.1 6.7 7.4 63.1 13.8 27 locomotion below pH 6.8, although some 5.4 7.8 64.4 20.9 24 cells moved slightly at the beginning of in5.2 8.2 65.8 17.3 26 cubation. Above pH 7.0, the locomotive speed increased with the rise in the pH value (table 2). persed into single cells. By means of shortIn order to exclude the possibility that the lived pseudopods, these cells migrated ac- behavior of the cells was affected by the tively in random direction on the substrate change in pH rather than by the charge of (fig. 2). the substrate, observations were made of On the BSA-lys substrate, the cells ad- cells on the BSA substrate, the BSA-lys hered immediately and spread into a flat- substrate and on the plastic substrate in metened shape. These cells ceased locomotion dium with various pH values ranging from by IO h of incubation (fig. 3). During their 6.6 to 8.2. On the BSA substrate, the avermitotic phase, the cells rounded up then age speed of cell locomotion after 1 day of spread again to become immobilized after incubation was 60-70 pm/h, and the decell division. On the plastic Petri dish, the cells also adhered immediately and spread, but the Table 4. Speed of cell locomotion on the shape of the spreading cells was different BSA-lys substrates as a function of pH from that on the BSA-lys substrate (fig. changes 4). This difference appeared to reflect the Average strength of the cell substrate adhesion. The AverNo. of duration of spreading cells moved actively in random cells measuring for age measeach cell speed directions. (pm/h) S.D. ured (hours) PH In spite of the diversity in cell behavior described above, there was only a little dif- First day of incubation 2.6 1.6 19 10.0 ference in the growth rates among the cells 6.6 7.0 3.1 2.7 32 9.6 grown on these three different types of sub- 7.4 I .5 1.1 33 10.0 7.8 6.2 4.0 :; 9.5 strates (fig. 5). 8.2 1.3 2.3 10.0 When the culture medium lacked serum, the cells scarcely migrated on any sub- Second day of incubation I .6 I.2 28 9.5 strate. Adhesion and spreading occurred in 6.6 7.0 0.5 0.6 3-i 10.0 a manner similar to that in the serum-con- 7.4 0.6 0.5 10.0 I.1 I.0 26 10.0 taining medium except that the cells never 7.8 8.2 0.9 1.4 28 9.8 adhered to the BSA substrate. Serum com-

250

Sugimoto and Hagiwara

Table 5. Speed of cell locomotion on the plastic substrates as a .function of pH changes No. of cells measured

Average duration of measuring for each cell (hours)

First day of incubation 39. I 11.3 6.6 49.0 19.2 7.0 39.1 17.9 7.4 39.3 9.7 7.8 29.8 10.9 8.2

14 II IS 15 IO

6.3 6.1 7.0 6.2 7.2

Swod 6.6 7.0 7.4 7.8 8.2

I6 I2 14 16 IO

8.4 5.3 6.2 6.7 9.2

I6 I5 I4 I2 I4

7.9 7.8 7.7 6.2 8.2

PH

Average speed &m/h)

S.D.

dny o$ incubation 27.2 4.5 31.6 7.9 29.6 7.0 27.4 15.8 27.8 6.7

Third dry c?fir?cuhntion 25.2 5.4 6.6 25.0 6.6 7.0 25.7 2.9 7.4 24.2 4.4 7.8 8.2 26.2 7.1

pendency on pH, as observed on the BSAhis substrate, was not seen (tables 2, 3). On the BSA-lys substrate, all the cells ceased locomotion independently of pH (table 4). Since the cell nucleus was tracked

for measuring cell mobility, the minor motion recorded for immobilized cells resulted only from the movement of the nucleus within the cell body. The cells with speed values below 5 pm/h, in fact, did not change their positions at all within the IO h period, except for the dispersal of two daughter cells to the neighborhood following mitosis. On the plastic substrate, the rate of locomotion was rather high at the beginning of incubation then slowed down with time, settling into a range of 20-30 pm/h independent of pH. This average speed compares well with that measured for the BSAhis substrate at pH 7.4 (table 5). Fig. 6 illustrates that the pH of the medium in the plastic dishes changed only slightly throughout the course of incubation. Effect of TNBS-treatment of substrates on cell locomotion To determine for certain whether the cells react to the charge of the substrate, the effect of the positively charged amino sites of the BSA-lys substrate on cell immobilization was studied. The results summarized in table 6 show that when this substrate was treated with TNBS, it no longer caused immobilization of the cells. Even if it was pre-

Table 6. Effect of TNBS treatment of substrates on cell locomotion” Cell locomotion No. of cells measured

Substrate

Preincubated with

TNBS Wml)

Average speed &m/h)

BSA-lys

MEM+S

50 0

30.5 I .6

7.9 I .7

13 22

BSA-lys

MEM

SO 0

27.6 I .8

5.4 I .4

I2 21

Plastic

MEM+S

50 0

42.5 41.2

7.4 9.7

I5 I3

% FCS

% FCS

S.D.

U 2-3x IO5 cells were seeded with IO ml of the assay medium buffered at pH 7.2 onto the serum-pretreated and TNBS-treated substrates, and incubated at 37°C. Cell locomotion was recorded cinematographically for the first 20 h of incubation. The speed of cell locomotion was measured as shown in Materials and Methods.

Substrate

charge

and cell locomotion

25 1

cubation with serum-containing medium. Thus the substrate charge is a main factor modulating cell behavior, although a serurn I’:2 component(s) may play an important role in cell movement. The immobilization that occurred on the 74-----L BSA-lys substrate was not due to poor cell 7.2. condition because growth continued at a 7.0-e high level (fig. 5) and active movement of the cytoplasmic inclusions and cell margin 68in the immobilized cells could be seen. 6.6-b They must have been essentially healthy. 2 3 0 1 It has been suggested that cell adhesion and Fig. 6. Abscissa: time (days); ordinate: pH. spreading are different on differently Changes in pH of the assay medium caused by cell charged substrates [18], and that cell mogrowth. L cells (3X IO5 cells) with 10 ml of the assay medium were seeded into the plastic dish and incubility depends on the strength of cell-subbated at 37°C. strate adhesion [2,3]. For this reason it may be considered that the immobilization seen on the less negatively charged substrates reincubated with serum-containing culture sulted because the cells stuck too tightly to medium before the TNBS treatment, the move on the substrates. cells migrated at similar speeds. In the case Electrophoretic mobilities were given as of the plastic substrate, however, the TNBS a relative value of the net surface charge treatment after preincubation with the se- because both zeta potential and surface rum-containing medium did not bring about charge density are directly proportional to any marked changes in cell behavior. the mobility. In the particle electrophore6.2

DISCUSSION In this study, a serum-containing medium was used because a serum component(s) was required for cell movement. Serum proteins were adsorbed to the substrate surface and might have modified its characteristic. The experimental results summarized in fig. 7, however, seem to show that cell locomotion varies depending on the substrate charge. Since TNBS is thought mainly to block the positively charged amino groups under the present experimental conditions as described in [17], the results in table 6 indicate that the positively charged amino sites on the BSA-lys substrates remained effective even after prein-

80

60

b.6

7.0

7.4

7.6

8.2

Fig. 7. Ahsrissn: pH; ordinure: speed of cell locomotion (pm/h). Cell locomotion as a function of pH on the BSA-his substrate (-), on the BSA substrate (---), on the BSAlys substrate (---) and on the plastic substrate (- -- -) after 24 h of incubation.

252

Sugimoto and Hagiwara

sis, immobilization occurred on a substrate having a surface negative charge of less than 53% of the BSA substrate and the change from immobilization to active locomotion was produced by a 25 % increase in the BSA-his substrate charge. These results suggest that cell locomotion can be easily effected by a slight change of surface charge at the cell contact area, and that cell movement occurring in vivo is also controlled by electrostatic interactions. In fact, another study [8] has reported that 46% increase in electrophoretic mobility occurs in gray crescent cells of the emphibian blastula as they progress to become the chordamesoderm. More than 90% of the total cell surface charges are anionic [ 193.Various cell functions such as endocytosis, membrane permeability, transmembrane potential [20, 211, cell movement and chemotaxis [22, 231 have been considered to be related to the anionic sites on the cell surfaces. It has been demonstrated that anionic sites are not evenly distributed around the cell surface and redistribution of the sites occurs with movement that is consistent with the fluid mosaic model of the plasma membrane [24, 251. Movement of some receptor sites on the cell surface is under the control of the cells’ intrinsic mechanism [26]. Cationic groups, however, also are present on the cell surface and play a demonstrable role in cell aggregation [27]. It can be assumed that cell surfaces bear the same electrostatic relationships to other cells as to the less negatively charged substrates. If this assumption is correct, the redistribution of differently charged sites on the cell surface

plays an important role in controlling various cell behaviors. The authors are grateful to Drs M. Yoneda and T. S. Okada for their helpful suggestions and observations and a critical reading of the manuscript. The authors also wish to thank Dr M. Takeichi for his helpful advice.

REFERENCES I. Curtis, A S G, The cell surface: its molecular role in morphogenesis, pp. 184-221. Logos/Academic Press, New York (1967). 2. Carter, S B, Nature 208 (1965) 1183. 3. Gail, M H & Boone, C W, Exp cell res 70 (1972) 33.

4. Weiss, L, Exp cell res 5 I (1968) 609. 5. Dan, K, Physiol zoo1 9 (1936) 43. 6. Garrod, D R & Gingell, D, J cell sci 6 (1970) 277. 7. Lee. K C. J cell sci 10 (1972) 249. 8. Schaeffer, B E, Schaeffer, H E & Brick, I, Dev biol 34 (1973) 66. 9. Macieira-Coelho, A & Avrameas, S, Proc natl acad sci US 69 (1972) 2469. IO. Oshima, N, Dev growth differ I7 (1975) 19. Il. Hebb, C R & Chu, M W, Exp cell res 20 (1960) 453.

12. Weiss, L& Kapes, DL, Expcell res4l (1966)601. 13. Lipton, A, Klinger, I, Paul, D & Halley, R W, Proc natl acad sci US 68 (1971) 2799. 14. Clarke, G D, Stoker, M G P, Ludlow, A & Thornton, M, Nature 227 (1970) 798. 15. Dulbecco, R, Nature 227 (1970) 802. 16. Gail, M H & Boone, C W, Exp cell res 64 (1971) 156. 17. Weiss, L, Exp cell res 83 (1974) 31I. 18. Takeichi, M & Okada, T S, Exp cell res 74 (1972) 51. 19. Mehrishi, J N, Prog biophys mol biol 25 (1972) I. 20. Brandt, P W & Freeman, A R, J colloid interface sci 25 (1967) 47. 21. Nagura, H,‘Asai, J, Katsumata, Y & Kojima, K, Acta pathol jap 23 (1973) 279. 22. Komnick, H, Stockem, W & Wohlfarth-Bottermann, K E, Int rev cytol 34 (1973) 169. 23. Wolpert, L & Gingell, b, Symp sot exp biol 22 (1968) 169. 24. Grinnell, F, Tobleman, M Q & Hackenbrock, C R, J cell biol 66 (1975) 470. 25. Weiss, L & Zeigel, R, J theor biol 34 (1972) 2 I. 26. Edelman, G M, Science 192(1976) 218. 27. Maslow, D E&Weiss, L, J cell sci 21 (1976) 219. Received April 22, 1977 Revised version received November 21, 1978