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The effects of antimicrotubule agents on cell motility in fibroblast aggregates
Pnnted I” Sweden Copyright @ 1979 by Academic Press. Inc. All right5 of reproduction in any form resrved 0014-4827/79/060359-06$02.00/U
Experimental Cell Research 120 (1979) 359-364
THE EFFECTS CELL
OF ANTIMICROTUBULE
MOTILITY
MARGARET Department
IN FIBROBLAST
AGENTS
ON
AGGREGATES
T. ARMSTRONG and PETER B. ARMSTRONG
ofZoology,
University
of California,
Davis, CA 95616, USA
SUMMARY Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in tibroblast motility in the three-dimensional aggregates.
In culture, fibroblast motility involves the protrusion of flattened cell processes or lamellipodia which characteristically are restricted to a limited portion of the cell’s perimeter [ 1, 31. In the absence of collision with other cells, the position of the leading lamella persists for considerable periods, ensuring that the cell moves in a relatively straight line [ 11, 261. During locomotion in culture, fibroblasts tend to become elongated in shape as the leading lamella advances over the substratum [l, 24, 261. Microtubules appear to be important for the restriction of lamellipodial activity to a limited portion of the cell’s margin and for the characteristic elongated spindle shape adopted by fibroblasts on two-dimensional surfaces since exposure to antimicrotubule agents results in an expansion of lamellipodial activity around the entire perimeter and a loss of the elongated shape [8, 12, 13, 16, 17, 25, 27, 281. There is a marked decrease in directed cell movement as displayed during wound healing [27, 281
and in the rate of emigration of cells from cellular aggregates adherent to a substrate
Bl. The involvement of microtubules in mediating the placement of lamellipodia has been demonstrated only for cells migrating in monolayers on artificial surfaces (usually glass or plastic). As an attempt to duplicate more exactly the in vivo situation of mesenchymal tissue, the present study assessed the effects of antimicrotubule agents on motility of fibroblasts within three-dimensional aggregates maintained in organ culture. Consistent with the observations on cells in monolayer culture, motility of tibroblasts in mesenchymal aggregates is significantly reduced by antimicrotubule agents, suggesting that microtubules do play a role in the control of cell movement in intact tissues. MATERIALS
AND METHODS
The methods used were as previously described for the analysis of cell movement in cell aggregates [4]. Exp CellRrs I20 (1979)
360
Armstrong
and Armstrong
Culture media and chemicals The culture medium was 9 parts Dulbecco-rr--‘:G-A IUULIIG” Eagle’s medium (Gibco powdered medium) plus 1 part heat-inactivated chicken serum (Gibco). Medium contained 100 U/ml penicillin, 100 pg/ml streptomycin and 1 pg/ml fungizone. The gas phase was 5% CO,, 95 % air. Cells were labelled during growth of the initial monolayers with [ Me-3H]thymidine (1 &i/ml). Antimicrotubule agents added to the medium at the time of fusion of the paired aggregates were: Colchicine (Sigma, 1OWM), Colcemid (CIBA, 1 pg/ml), Vinblastine sulfate (Sigma, 10-OM), and R17934 (nocodazole, AI>-f-L f -1-I >:I__.a> L---- - C i-l-1 -‘en,k so,ution in DM&) [8]. Cultures exposed co the antitubule agents were shielded from light to avoid photoinactivation of the drug.
Fibroblast
aggregates
Primary monolayer cultures of tibroblasts were prepared from trypsin-dispersed IO-day chick embryo heart ventricle, back skin and sclera. The sclera was separated from the pigment epithelium following treatment of IO-day chick embryo eyes in 3 % Difco 1: 250 trypsin dissolved in Ca*+-, Mgz+-free saline (37”C, pH 7.3, 35 min). Cells were plated at 12-20X106 cells in 90 mm Falcon tissue culture dishes. Non-fibroblastic heart and skin cells were removed by decanting the unattached cells after 0.5-1.5 h at 37°C as suggested by Polinger [19]. Fresh culture medium containing [3H]thymidine was added and the cultures were grown to confluency (requiring 2-3 days). The confluent monolayer was then scored with a rubber policeman to divide it into squares 0.5-l .O cm per side. The pieces were gently scraped from the dish and were allowed to round un into compact spherical aggregates by overnight culture in shaker Basks (gyratory shaker, 25 ml flask, 90 rpm, 37°C). Shaker flasks were gassed with 5 % CO*, 95 % air, then sealed with silicon rubber stoppers (Bellco, Vineland, N.J.).
Heart myocyte aggregates Suspensions of heart myocytes freed of most of the contaminating mesenchymal cells were obtained as the cells which did not attach when dispersed lo-day chick embryo heart ventricle cell populations were incubated in 90-mm Falcon tissue culture plastic dishes at 37°C for 1.5 h. Coherent mvocvte tissue was orenared bv pelleting the cells by gentle centrifugatibn -in 16ml screw-car, test tubes (Pyrex No. 9826). followed by stationary incubation for 3-6 h at 37°C. The coherent pellets were removed and chopped into smaller aggreeates with small knives made from sewina needles. yhe aggregates were then allowed to round-up during an incubation overnight at 37°C in 25 ml Ehrlenmeyer flasks placed on a gyratory shaker.
Fusion of pairs of aggregates Pairs of spherical aggregates (one labelled, one unlabelled) were placed in hanging drops of culture medium [2, 181to which the antimicrotubule agent was added or omitted in the control experiments. The agExp CdRes
120(1979)
aregates came into contact at the bottom of the hanakg-drop and wit hin 3 to 6 h adhered tightly enough to enable them to be removed and transferred to shaker flasks for further culture. The antimicrotubule agents had no apparent effect on the adhesion of the aggregates. The antimicrotubule agents were added to the medium in the shaker flasks in the appropriate cases. The culture medium was supplemented with unlabelled thymidine (1 wM/ml) for rounding up of both labelled and unlabelled monolayer aggregates for hanging drops, and for subsequent aggregate pairs. This reduced the incorporation into unlabelled cells of labelled thymidine transferred from labelled cells.
Histology-_ and measurement Aggregates to be used for light microscopy and autoradioeraohv were fixed in Bouin’s fixative. embedded in paFa&*and sectioned at 5 I.crn.During embedding, the aggregate pairs were oriented in the paraffin block so that the plane of sectioning would pass approximately perpendicular to the plane of fusion of labelled and unlabelled aggregate. Proper orientation was easily determined since most aggregate pairs were still ellipsoidal at the time of fixation. All aggregates were serially sectioned, so any aggregate pairs that were not so oriented could be readily identified and were excluded from our analysis. Measurements of the distances of cell migration in aggregates were made on the sectioned, paraffin-embedded material. For each aggregate pair, the section with the widest diameter was taken to be the section passing through the middle of the aggregate pair (the “mid-sagittal section”). Measurements of the distances moved by labelled cells into the unlabelled aggregate were made for each pair on a total of 10 sections centered on the mid-sagittal region, with one or two sections between each used for measurement to ensure that different cells were seen each time. The distance of migration was taken as the distance between the
Fig. I. Autoradiogram of [3H]thymidine-labelled heart fibroblast aggregate cultured in hanging-drop culture in contact with an unlabelled heart fibroblast aggregate for 2 days. Labelled cells are distinguished by black silver grains lying over their nuclei. The arrows indicate labelled cells that have moved into the peripheral zone of the unlabelled aggregate. X 133. Fig. 2. Colchicine-treated heart libroblast aggregate pair fused for 2 days. The arrow marks the only labelled cell that has moved into the unlabelled aggregate. X 133. Fig. 3. [3H]thymidine-labelled heart tibroblast aggregate fused with an unlabelled heart myocyte aggregate for 2 days. The myocyte aggregate has spread around the fibroblast aggregate and individual fibroblasts have invaded the rnf&y& aggregate (arrows). x 133. Fk. 4. Colcemid-treated 13Hlthvmidine-labelled heart fibroblast aggregate fused with an unlabelled heart myocyte aggregate for 2 days. There is no spreading of the myocyte aggregate around the tibroblast aggregate. The arrows indicate tibroblasts that have invaded a short distance into the myocyte aggregate. x 123.
Motility in fibroblast aggregates
361
Exp CellRcs 120(1979)
362
Armstrong
and Armstrong
Table 1. Average maximum cell movement in paired heartfibroblast aggregatesa
Media
No. of days in culture*
No. of paired aggregates
pm
Control Colcemid Vinblastine R17934 Control Colcemid Vinblastine R17934 Colchicine
1 1 I 1 2 2 2 2 2
8 11 10 9 11 10 7 3 6
85.Ok16.8’ 10.2+ 5.9 11.1+ 9.0 14.9k 7.2 249.1k72.3 26.9+ 9.0 37.0f 8.4 38.9f 4.3 58.6516.8
u Average of 10 measurements of the maximum distance of cell movement made on each one of the naired aggregates. b Number of days the labelled and unlabelled aggregates have been in opposition. c S.E.M.
plane of fusion of the two aggregates (determined by the plane in the interior of the aggregate where little mixing occurred: see Results) and the furthermost labelled cell in the unlabelled aggregate. Measurements were performed with an ocular micrometer previously calibrated with a stage micrometer.
RESULTS Cell motility in solid tissue masses was assessed by the extent of migration of cells from the labelled member of a fused pair of cell aggregates into the unlabelled aggregate. In experimental aggregate pairs, an antimicrotubule agent (either colchicine, colcemid, vinblastine or R17934) was present in the medium from the time of aggregate fusion. Homotypic
heartfibroblast
aggregate pairs
When placed in contact, heart fibroblast aggregates adhered to each other and rapidly broadened the area of mutual contact. Control heart fibroblast aggregates had two zones: a peripheral zone 3-8 cells thick consisting of flattened cells, and an interior reErp Cd Res I20 (1979)
gion of stellate, more loosely packed cells. In control aggregates, the peripheral cells were motile, with considerable intermingling of labelled and unlabelled cells being apparent within 1 day; whereas the internal stellate cells were nearly stationary (fig. 1). In aggregates treated with antimicrotubule agents there was a reduction in the number of flattened cells in the peripheral zone and cell motility was greatly reduced. The motility that did occur was still restricted to the peripheral zone of the aggregate (figs 1, 2; table 1). The average maximum distance moved by labelled cells into the unlabelled aggregate in aggregate pairs treated with an antimicrotubule agent and fused for 1 day was 12.Ok7.5 pm while that in control aggregate pairs was 85.0+16.8 pm. In aggregate pairs fused for 2 days the average maximum distances moved were 38.3? 16.0 pm (treated) and 249.lk72.3 pm (controls). Homotypic skin and sclera fibroblast aggregate pairs
In control homotypic skin and sclera fibroblast aggregate pairs, cell motility occurred both in the interiors and in the peripheries of the aggregates. Elongated cells were present in both regions. Migratory cells were frequently organized as lines or sheets of elongated cells. Exposure to antimicrotubule agents reduced the intermingling of labelled and unlabelled cells in the aggregate pairs with a concomitant reduction in the numbers of elongated cells. Heterotypic heart fibroblast heart myocyte aggregate pairs
When a heart myocyte aggregate was fused with an aggregate of [3H]thymidine-labelled fibroblasts in control medium, the two cell types showed different patterns of behavior. The myocytes spread around the libroblast aggregate as a coherent sheet and the
Motility in fibroblast aggregates fibroblasts invaded the myocyte aggregate as individual cells (fig. 3). When an antimicrotubule agent was added at the time of fusion of the aggregates, the spreading of the myocytes around the tibroblast aggregate was completely abolished and the extent of invasion of the myocyte aggregate by labelled fibroblasts was reduced (fig. 4). DISCUSSION The morphology and motility of tissue cells involves the several elements of the cytoskeleton (the microfilaments, intermediate filaments and microtubules) [2, 141.The use of drugs that selectively depolymerize one or another of the cytoskeletal components has proven useful in unraveling their respective functions [7, 201. Vertebrate fibroblasts maintained in monolayer tissue culture show characteristic alterations in form and movement upon exposure to antimicrotubule agents [8, 12, 13, 16, 17, 25, 27, 281. Lamellipodial extension and retraction continues, indicating that microtubules are not involved in these processes. The cells do, however, lose their elongated shape and the leading lamella which, in untreated cells is restricted to a limited portion of the cell’s perimeter, broadens until it encompasses most or all of the cell’s border. In this condition, net cell translocation is markedly diminished [12]. Based largely on these inhibitor studies, it has been suggested that microtubules function to maintain cell polarity by stabilizing the leading lamella, restricting lamellipodial activity to a limited portion of the cell’s margin. To date, studies of this nature have been limited to cells maintained in two-dimensional arrays adhering to glass or plastic surfaces, which is admittedly an artificial situation for mesenchymal cells. The present study examined the motility of cells cul-
363
tured in three-dimensional aggregates as reflecting more closely the organization of mesenchymal tissue in vivo. We observed that pharmacological concentrations of the antimicrotubule agents colcemid, vinblastine, colchicine and R17934 (nocodazole) produced a marked decrease in the migration of mesenchymal cells in cell aggregates. In heart fibroblast aggregates, the average maximal movement was reduced from 85.Ok16.8 pm in 1 day (controls) to 12.0&-7.5pm in 1 day, an 86% decrease. In mesenchymal aggregates of heart, sclera and skin fibroblasts, the cells which migrated appeared predominantly to be flattened or elongated in form. A similar correlation of motility and elongation has been observed in dense monolayers [9, lo] and in intercellular invasion by Iibroblasts in vivo [22]. Concomitant with the decrease in cell movement, the antimicrotubule agents produced a decrease in the frequency of the elongated shape. When aggregates of dissimilar cells are paired, cells of one type usually migrate over the surface of the partner aggregate to envelop it [5, 6, 15, 21, 231. In the present study, antimicrotubule agents prevented the spreading of cardiac myocytes over the surfaces of Iibroblast aggregate partners. This study was supported by NSF grant PCM 77. 18950 and Cancer Research Funds of the University of California. Note added in prooj: The present report notes that invasion of heart myocyte aggregates by fibroblasts is inhibited by antimicrotubule agents. In a recent study, Marcel & De Brabander (Marcel, M M K & De Brabander, M, J nat cancer inst 61 (1978) 787) report that microtubule inhibitors suppress invasion of tumor cells into embryonic chick heart fragments in organ culture.
REFERENCES 1. Abercrombie, M, Exp cell res, suppl. 8 (1961) 188. 2. - Ciba foundation symposium on cell motility. Elsevier, Amsterdam (1973). 3. Ambrose, E J, Exp cell res, suppl. 8 (1961) 54. E-up Cell Rcs I20 (1979)
364
Armstrong
and Armstrong
Armstrong, M T & Armstrong, P B, J cell sci
19. Polinger, I S, Exp cell res 76 (1973) 243.
33 (19781 37.
20. Soifer, D, The biology of cytoplasmic
Armstrong, PB, Dev bio164 (1978) 60. Armstrong. P B & Niederman. , R. Dev biol 28 (1972) 51c’ Borgers, M & De Brabander, M, Microtubules and microtubule inhibitors. North-Holland, Amsterdam (1975). 8. De Brabander, M J, Van de Veire, R M L, Aerts. F E M, Borgers, M & Janssen, P A J, Cancer res 36 (1976) 905. 9. Elsdale, T R, Exp cell res 51 (1968) 439. 10. Elsdale, T R & Bard, J, Nature 236 (1972) 152. 11. Gail, M H &Boone, C W, Biophys j 10(1970) 980. 12. - Exp cell res 65 (1971) 221. 13. Goldman, R D, J cell bio151 (1971) 752. 14. Goldman, R, Pollard, T & Rosenbaum, J, Cell motilitv. Cold Sorina Harbor conference on all prolife%ion, vol: 3. told Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1976). 15. Holtfreter, J, Z exp zellforsch 23 (1939) 169. 16. Ivanova. 0 Y. Mareolis. L B. Vasiliev. J M & Gelfand; I M, Exp cgll res 101 (1976) 207.’ 17. Miszurski, B, Exp cell res, suppl. 1 (1949) 450, 18. Niederman, R & Armstrong, P B, J exp zoo1 181 (1972) 17.
Exp CellRes IZO(1979)
microtubules. New York Academy of Science. New York (1975). 21. Steinberg, M S, J exp zoo1 173 (1970) 395. 22. Tickle. C. Crawlev. A & Goodman. M. J cell sci 31 (1978)293. zoo1 128 (1955) 23. Townes, P & Holtfreter, 53. 24. Trinkaus, J P, Bethchaku, T & Krulikowski, 25. 26. 27. 28.
L S, Exp cell res 64 (1971) 291. Van der Schueren, B, Cassiman, J J & Van den Berghe. H, J cell sci 31 (1978) 355. Vasiliev, J M & Gelfand, I M, Fundamental aspects of metastasis (ed L Weiss) p. 71. NorthHolland, Amsterdam (1976). Vasiliev, J M, Gelfand, I M, Domnina, L V, Ivanova, 0 Y, Komm, S G & Olshevskaja, L V, J embryo1 exp morph01 24 (1970) 625. Vasiliev, J M, Gelfand, I M, Domnina, L V & Rapport, R I, Exp cell res 54 (1969) 83.
Received October 23, 1978 Accepted December 20, 1978
Experimental Cell Research 120 (1979) 359-364
THE EFFECTS CELL
OF ANTIMICROTUBULE
MOTILITY
MARGARET Department
IN FIBROBLAST
AGENTS
ON
AGGREGATES
T. ARMSTRONG and PETER B. ARMSTRONG
ofZoology,
University
of California,
Davis, CA 95616, USA
SUMMARY Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in tibroblast motility in the three-dimensional aggregates.
In culture, fibroblast motility involves the protrusion of flattened cell processes or lamellipodia which characteristically are restricted to a limited portion of the cell’s perimeter [ 1, 31. In the absence of collision with other cells, the position of the leading lamella persists for considerable periods, ensuring that the cell moves in a relatively straight line [ 11, 261. During locomotion in culture, fibroblasts tend to become elongated in shape as the leading lamella advances over the substratum [l, 24, 261. Microtubules appear to be important for the restriction of lamellipodial activity to a limited portion of the cell’s margin and for the characteristic elongated spindle shape adopted by fibroblasts on two-dimensional surfaces since exposure to antimicrotubule agents results in an expansion of lamellipodial activity around the entire perimeter and a loss of the elongated shape [8, 12, 13, 16, 17, 25, 27, 281. There is a marked decrease in directed cell movement as displayed during wound healing [27, 281
and in the rate of emigration of cells from cellular aggregates adherent to a substrate
Bl. The involvement of microtubules in mediating the placement of lamellipodia has been demonstrated only for cells migrating in monolayers on artificial surfaces (usually glass or plastic). As an attempt to duplicate more exactly the in vivo situation of mesenchymal tissue, the present study assessed the effects of antimicrotubule agents on motility of fibroblasts within three-dimensional aggregates maintained in organ culture. Consistent with the observations on cells in monolayer culture, motility of tibroblasts in mesenchymal aggregates is significantly reduced by antimicrotubule agents, suggesting that microtubules do play a role in the control of cell movement in intact tissues. MATERIALS
AND METHODS
The methods used were as previously described for the analysis of cell movement in cell aggregates [4]. Exp CellRrs I20 (1979)
360
Armstrong
and Armstrong
Culture media and chemicals The culture medium was 9 parts Dulbecco-rr--‘:G-A IUULIIG” Eagle’s medium (Gibco powdered medium) plus 1 part heat-inactivated chicken serum (Gibco). Medium contained 100 U/ml penicillin, 100 pg/ml streptomycin and 1 pg/ml fungizone. The gas phase was 5% CO,, 95 % air. Cells were labelled during growth of the initial monolayers with [ Me-3H]thymidine (1 &i/ml). Antimicrotubule agents added to the medium at the time of fusion of the paired aggregates were: Colchicine (Sigma, 1OWM), Colcemid (CIBA, 1 pg/ml), Vinblastine sulfate (Sigma, 10-OM), and R17934 (nocodazole, AI>-f-L f -1-I >:I__.a> L---- - C i-l-1 -‘en,k so,ution in DM&) [8]. Cultures exposed co the antitubule agents were shielded from light to avoid photoinactivation of the drug.
Fibroblast
aggregates
Primary monolayer cultures of tibroblasts were prepared from trypsin-dispersed IO-day chick embryo heart ventricle, back skin and sclera. The sclera was separated from the pigment epithelium following treatment of IO-day chick embryo eyes in 3 % Difco 1: 250 trypsin dissolved in Ca*+-, Mgz+-free saline (37”C, pH 7.3, 35 min). Cells were plated at 12-20X106 cells in 90 mm Falcon tissue culture dishes. Non-fibroblastic heart and skin cells were removed by decanting the unattached cells after 0.5-1.5 h at 37°C as suggested by Polinger [19]. Fresh culture medium containing [3H]thymidine was added and the cultures were grown to confluency (requiring 2-3 days). The confluent monolayer was then scored with a rubber policeman to divide it into squares 0.5-l .O cm per side. The pieces were gently scraped from the dish and were allowed to round un into compact spherical aggregates by overnight culture in shaker Basks (gyratory shaker, 25 ml flask, 90 rpm, 37°C). Shaker flasks were gassed with 5 % CO*, 95 % air, then sealed with silicon rubber stoppers (Bellco, Vineland, N.J.).
Heart myocyte aggregates Suspensions of heart myocytes freed of most of the contaminating mesenchymal cells were obtained as the cells which did not attach when dispersed lo-day chick embryo heart ventricle cell populations were incubated in 90-mm Falcon tissue culture plastic dishes at 37°C for 1.5 h. Coherent mvocvte tissue was orenared bv pelleting the cells by gentle centrifugatibn -in 16ml screw-car, test tubes (Pyrex No. 9826). followed by stationary incubation for 3-6 h at 37°C. The coherent pellets were removed and chopped into smaller aggreeates with small knives made from sewina needles. yhe aggregates were then allowed to round-up during an incubation overnight at 37°C in 25 ml Ehrlenmeyer flasks placed on a gyratory shaker.
Fusion of pairs of aggregates Pairs of spherical aggregates (one labelled, one unlabelled) were placed in hanging drops of culture medium [2, 181to which the antimicrotubule agent was added or omitted in the control experiments. The agExp CdRes
120(1979)
aregates came into contact at the bottom of the hanakg-drop and wit hin 3 to 6 h adhered tightly enough to enable them to be removed and transferred to shaker flasks for further culture. The antimicrotubule agents had no apparent effect on the adhesion of the aggregates. The antimicrotubule agents were added to the medium in the shaker flasks in the appropriate cases. The culture medium was supplemented with unlabelled thymidine (1 wM/ml) for rounding up of both labelled and unlabelled monolayer aggregates for hanging drops, and for subsequent aggregate pairs. This reduced the incorporation into unlabelled cells of labelled thymidine transferred from labelled cells.
Histology-_ and measurement Aggregates to be used for light microscopy and autoradioeraohv were fixed in Bouin’s fixative. embedded in paFa&*and sectioned at 5 I.crn.During embedding, the aggregate pairs were oriented in the paraffin block so that the plane of sectioning would pass approximately perpendicular to the plane of fusion of labelled and unlabelled aggregate. Proper orientation was easily determined since most aggregate pairs were still ellipsoidal at the time of fixation. All aggregates were serially sectioned, so any aggregate pairs that were not so oriented could be readily identified and were excluded from our analysis. Measurements of the distances of cell migration in aggregates were made on the sectioned, paraffin-embedded material. For each aggregate pair, the section with the widest diameter was taken to be the section passing through the middle of the aggregate pair (the “mid-sagittal section”). Measurements of the distances moved by labelled cells into the unlabelled aggregate were made for each pair on a total of 10 sections centered on the mid-sagittal region, with one or two sections between each used for measurement to ensure that different cells were seen each time. The distance of migration was taken as the distance between the
Fig. I. Autoradiogram of [3H]thymidine-labelled heart fibroblast aggregate cultured in hanging-drop culture in contact with an unlabelled heart fibroblast aggregate for 2 days. Labelled cells are distinguished by black silver grains lying over their nuclei. The arrows indicate labelled cells that have moved into the peripheral zone of the unlabelled aggregate. X 133. Fig. 2. Colchicine-treated heart libroblast aggregate pair fused for 2 days. The arrow marks the only labelled cell that has moved into the unlabelled aggregate. X 133. Fig. 3. [3H]thymidine-labelled heart tibroblast aggregate fused with an unlabelled heart myocyte aggregate for 2 days. The myocyte aggregate has spread around the fibroblast aggregate and individual fibroblasts have invaded the rnf&y& aggregate (arrows). x 133. Fk. 4. Colcemid-treated 13Hlthvmidine-labelled heart fibroblast aggregate fused with an unlabelled heart myocyte aggregate for 2 days. There is no spreading of the myocyte aggregate around the tibroblast aggregate. The arrows indicate tibroblasts that have invaded a short distance into the myocyte aggregate. x 123.
Motility in fibroblast aggregates
361
Exp CellRcs 120(1979)
362
Armstrong
and Armstrong
Table 1. Average maximum cell movement in paired heartfibroblast aggregatesa
Media
No. of days in culture*
No. of paired aggregates
pm
Control Colcemid Vinblastine R17934 Control Colcemid Vinblastine R17934 Colchicine
1 1 I 1 2 2 2 2 2
8 11 10 9 11 10 7 3 6
85.Ok16.8’ 10.2+ 5.9 11.1+ 9.0 14.9k 7.2 249.1k72.3 26.9+ 9.0 37.0f 8.4 38.9f 4.3 58.6516.8
u Average of 10 measurements of the maximum distance of cell movement made on each one of the naired aggregates. b Number of days the labelled and unlabelled aggregates have been in opposition. c S.E.M.
plane of fusion of the two aggregates (determined by the plane in the interior of the aggregate where little mixing occurred: see Results) and the furthermost labelled cell in the unlabelled aggregate. Measurements were performed with an ocular micrometer previously calibrated with a stage micrometer.
RESULTS Cell motility in solid tissue masses was assessed by the extent of migration of cells from the labelled member of a fused pair of cell aggregates into the unlabelled aggregate. In experimental aggregate pairs, an antimicrotubule agent (either colchicine, colcemid, vinblastine or R17934) was present in the medium from the time of aggregate fusion. Homotypic
heartfibroblast
aggregate pairs
When placed in contact, heart fibroblast aggregates adhered to each other and rapidly broadened the area of mutual contact. Control heart fibroblast aggregates had two zones: a peripheral zone 3-8 cells thick consisting of flattened cells, and an interior reErp Cd Res I20 (1979)
gion of stellate, more loosely packed cells. In control aggregates, the peripheral cells were motile, with considerable intermingling of labelled and unlabelled cells being apparent within 1 day; whereas the internal stellate cells were nearly stationary (fig. 1). In aggregates treated with antimicrotubule agents there was a reduction in the number of flattened cells in the peripheral zone and cell motility was greatly reduced. The motility that did occur was still restricted to the peripheral zone of the aggregate (figs 1, 2; table 1). The average maximum distance moved by labelled cells into the unlabelled aggregate in aggregate pairs treated with an antimicrotubule agent and fused for 1 day was 12.Ok7.5 pm while that in control aggregate pairs was 85.0+16.8 pm. In aggregate pairs fused for 2 days the average maximum distances moved were 38.3? 16.0 pm (treated) and 249.lk72.3 pm (controls). Homotypic skin and sclera fibroblast aggregate pairs
In control homotypic skin and sclera fibroblast aggregate pairs, cell motility occurred both in the interiors and in the peripheries of the aggregates. Elongated cells were present in both regions. Migratory cells were frequently organized as lines or sheets of elongated cells. Exposure to antimicrotubule agents reduced the intermingling of labelled and unlabelled cells in the aggregate pairs with a concomitant reduction in the numbers of elongated cells. Heterotypic heart fibroblast heart myocyte aggregate pairs
When a heart myocyte aggregate was fused with an aggregate of [3H]thymidine-labelled fibroblasts in control medium, the two cell types showed different patterns of behavior. The myocytes spread around the libroblast aggregate as a coherent sheet and the
Motility in fibroblast aggregates fibroblasts invaded the myocyte aggregate as individual cells (fig. 3). When an antimicrotubule agent was added at the time of fusion of the aggregates, the spreading of the myocytes around the tibroblast aggregate was completely abolished and the extent of invasion of the myocyte aggregate by labelled fibroblasts was reduced (fig. 4). DISCUSSION The morphology and motility of tissue cells involves the several elements of the cytoskeleton (the microfilaments, intermediate filaments and microtubules) [2, 141.The use of drugs that selectively depolymerize one or another of the cytoskeletal components has proven useful in unraveling their respective functions [7, 201. Vertebrate fibroblasts maintained in monolayer tissue culture show characteristic alterations in form and movement upon exposure to antimicrotubule agents [8, 12, 13, 16, 17, 25, 27, 281. Lamellipodial extension and retraction continues, indicating that microtubules are not involved in these processes. The cells do, however, lose their elongated shape and the leading lamella which, in untreated cells is restricted to a limited portion of the cell’s perimeter, broadens until it encompasses most or all of the cell’s border. In this condition, net cell translocation is markedly diminished [12]. Based largely on these inhibitor studies, it has been suggested that microtubules function to maintain cell polarity by stabilizing the leading lamella, restricting lamellipodial activity to a limited portion of the cell’s margin. To date, studies of this nature have been limited to cells maintained in two-dimensional arrays adhering to glass or plastic surfaces, which is admittedly an artificial situation for mesenchymal cells. The present study examined the motility of cells cul-
363
tured in three-dimensional aggregates as reflecting more closely the organization of mesenchymal tissue in vivo. We observed that pharmacological concentrations of the antimicrotubule agents colcemid, vinblastine, colchicine and R17934 (nocodazole) produced a marked decrease in the migration of mesenchymal cells in cell aggregates. In heart fibroblast aggregates, the average maximal movement was reduced from 85.Ok16.8 pm in 1 day (controls) to 12.0&-7.5pm in 1 day, an 86% decrease. In mesenchymal aggregates of heart, sclera and skin fibroblasts, the cells which migrated appeared predominantly to be flattened or elongated in form. A similar correlation of motility and elongation has been observed in dense monolayers [9, lo] and in intercellular invasion by Iibroblasts in vivo [22]. Concomitant with the decrease in cell movement, the antimicrotubule agents produced a decrease in the frequency of the elongated shape. When aggregates of dissimilar cells are paired, cells of one type usually migrate over the surface of the partner aggregate to envelop it [5, 6, 15, 21, 231. In the present study, antimicrotubule agents prevented the spreading of cardiac myocytes over the surfaces of Iibroblast aggregate partners. This study was supported by NSF grant PCM 77. 18950 and Cancer Research Funds of the University of California. Note added in prooj: The present report notes that invasion of heart myocyte aggregates by fibroblasts is inhibited by antimicrotubule agents. In a recent study, Marcel & De Brabander (Marcel, M M K & De Brabander, M, J nat cancer inst 61 (1978) 787) report that microtubule inhibitors suppress invasion of tumor cells into embryonic chick heart fragments in organ culture.
REFERENCES 1. Abercrombie, M, Exp cell res, suppl. 8 (1961) 188. 2. - Ciba foundation symposium on cell motility. Elsevier, Amsterdam (1973). 3. Ambrose, E J, Exp cell res, suppl. 8 (1961) 54. E-up Cell Rcs I20 (1979)
364
Armstrong
and Armstrong
Armstrong, M T & Armstrong, P B, J cell sci
19. Polinger, I S, Exp cell res 76 (1973) 243.
33 (19781 37.
20. Soifer, D, The biology of cytoplasmic
Armstrong, PB, Dev bio164 (1978) 60. Armstrong. P B & Niederman. , R. Dev biol 28 (1972) 51c’ Borgers, M & De Brabander, M, Microtubules and microtubule inhibitors. North-Holland, Amsterdam (1975). 8. De Brabander, M J, Van de Veire, R M L, Aerts. F E M, Borgers, M & Janssen, P A J, Cancer res 36 (1976) 905. 9. Elsdale, T R, Exp cell res 51 (1968) 439. 10. Elsdale, T R & Bard, J, Nature 236 (1972) 152. 11. Gail, M H &Boone, C W, Biophys j 10(1970) 980. 12. - Exp cell res 65 (1971) 221. 13. Goldman, R D, J cell bio151 (1971) 752. 14. Goldman, R, Pollard, T & Rosenbaum, J, Cell motilitv. Cold Sorina Harbor conference on all prolife%ion, vol: 3. told Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1976). 15. Holtfreter, J, Z exp zellforsch 23 (1939) 169. 16. Ivanova. 0 Y. Mareolis. L B. Vasiliev. J M & Gelfand; I M, Exp cgll res 101 (1976) 207.’ 17. Miszurski, B, Exp cell res, suppl. 1 (1949) 450, 18. Niederman, R & Armstrong, P B, J exp zoo1 181 (1972) 17.
Exp CellRes IZO(1979)
microtubules. New York Academy of Science. New York (1975). 21. Steinberg, M S, J exp zoo1 173 (1970) 395. 22. Tickle. C. Crawlev. A & Goodman. M. J cell sci 31 (1978)293. zoo1 128 (1955) 23. Townes, P & Holtfreter, 53. 24. Trinkaus, J P, Bethchaku, T & Krulikowski, 25. 26. 27. 28.
L S, Exp cell res 64 (1971) 291. Van der Schueren, B, Cassiman, J J & Van den Berghe. H, J cell sci 31 (1978) 355. Vasiliev, J M & Gelfand, I M, Fundamental aspects of metastasis (ed L Weiss) p. 71. NorthHolland, Amsterdam (1976). Vasiliev, J M, Gelfand, I M, Domnina, L V, Ivanova, 0 Y, Komm, S G & Olshevskaja, L V, J embryo1 exp morph01 24 (1970) 625. Vasiliev, J M, Gelfand, I M, Domnina, L V & Rapport, R I, Exp cell res 54 (1969) 83.
Received October 23, 1978 Accepted December 20, 1978