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Role of fibronectin in the organisation of the cytoskeleton during the sreading of rat mammary epithelial cells
CeflBm~gylnternationalRepor~ VoLI~ No.~ 1992 ROLE
OF
FIBRONECTIN
CYTOSKELETON
Sharon
A.
DURING
Ferns, and
Department
IN THE
THE
ORGANISATION
SPREADING
EPITHELIAL
CELLS
Rosemary
Kimbell,
Michael
J.
of Histopathology,
Medical School,
207
OF
OF
RAT
Jane
A.
THE
MAMMARY
Aitken
Warburton.
St George's
London SWI7 ORE,
Hospital
UK.
ABSTRACT
The c o r r e l a t i o n between the e x t r a c e l l u l a r d e p o s i t i o n of f i b r o n e c t i n and the d e v e l o p m e n t of the a c t i n - c o n t a i n i n g cytoskeleton was studied during the a t t a c h m e n t and s p r e a d i n g of the rat m a m m a r y e p i t h e l i a l cell line Rama 25. D u r i n g the initial phase of cell spreading, actin is l o c a l i s e d in p e r i p h e r a l m i c r o f i l a m e n t bundles. As cell spreading .increases, the peripheral ring is d i s p l a c e d towards the p e r i n u c l e a r region. Fibronectin, d e p o s i t e d b e n e a t h the basal surface, c o - l o c a l i s e s with the a c t i n - c o n t a i n i n g p e r i p h e r a l ring. The p e r i p h e r a l ring s u b s e q u e n t l y disappears and is replaced by a system of radial microfilaments that extend from the perinuclear r e g i o n to the cell p e r i p h e r y . At this stage, there is no correlation b e t w e e n the d i s t r i b u t i o n of f i b r o n e c t i n and actin. As cells f o r m colonies, radial m i c r o f i l a m e n t bundles are r e p l a c e d by p e r i p h e r a l microfilament b u n d l e s w h i c h do not c o - l o c a l i s e with fibronectin. Cells at the edges of c o l o n i e s e x t e n d lamellae that contain m i c r o f i l a m e n t stress fibres. In these structures t h e r e is c o - l o c a l i s a t i o n of actin, fibronectin and the a551-integrin fibronectin receptor. INTRODUCTION
The functional d i f f e r e n t i a t i o n of mammary cells in v i t r o is dependent on the composition of the s u b s t r a t u m (Wicha et al, 1982). The a b i l i t y of m a m m a r y e p i t h e l i a l cells to s y n t h e s i s e milk p r o t e i n s in r e s p o n s e to l a c t o g e n i c hormones is m a x i m i s e d by g r o w t h on s u b s t r a t a (eg. r e c o n s t i t u t e d b a s e m e n t m e m b r a n e gels) that a l l o w cells to p o l a r i s e and form duct-like structure (Barcellos-Hoff et al, 1989). The e x t r a c e l l u l a r m a t r i x influences cell behaviour partly through binding to cell surface r e c e p t o r s whose c y t o p l a s m i c d o m a i n s interact with the c y t o s k e l e t o n (Albelda and Buck 1990). Evidence has been
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© 1992 Academic Press Ltd
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Cell Biology International Reports, VoL 16, No. 3, 1992
p r e s e n t e d that the i n t e g r i t y of the c y t o s k e l e t o n is r e q u i r e d for the h o r m o n a l i n d u c t i o n of m i l k p r o t e i n synthesis (Seely and A g g e l e r 1991). The c y t o s k e l e t o n also p a r t l y d e t e r m i n e s cell shape, g r o w t h c o n t r o l and the e x p r e s s i o n of the m a l i g n a n t p h e n o t y p e (Ben-Ze'ev 1985). D u r i n g transformation, changes in the d i s t r i b u t i o n of e x t r a c e l l u l a r matrix components and t h e i r r e c e p t o r s are a c c o m p a n i e d by a r e a r r a n g e m e n t of the cytoskeleton. The structure of the c y t o s k e l e t o n may be further m o d i f i e d by t y r o s i n e kinases for w h i c h several c y t o s k e l e t a l substrates exist (Glenney and Zokas 1989). Interactions of tumour cells with c h e m o t a c t i c or m o t i l ity factors (Watanabe et al, 1991) p r e s u m a b l y i n v o l v e the m i c r o f i l a m e n t system. Whereas considerable progress has been made in u n d e r s t a n d i n g microfilament and extracellular matrix interactions in normal and transformed fibroblasts, relatively little is k n o w n about t h e s e p r o c e s s e s in e p i t h e l i a l cells. We n o w report on the r e l a t i o n s h i p between e x t r a c e l l u l a r m a t r i x d e p o s i t i o n and m i c r o f i l a m e n t o r g a n i s a t i o n d u r i n g the a t t a c h m e n t and s p r e a d i n g of a rat m a m m a r y e p i t h e l i a l cell line. EXPERIMENTAL
PROCEDURES
Tissue Culture. Rama 25 cells were cultured in D u l b e c c o ' s m o d i f i e d Eagles m e d i u m s u p p l e m e n t e d with 5% fetal calf serum, 5 0 n g / m l h y d r o c o r t i s o n e and 5 0 n g / m l insulin. This cell line was isolated from a d i m e t h y l b e n z a n t h r a c e n e - i n d u c e d rat m a m m a r y t u m o u r (Bennett et al, 1978). Immunofluorescence Techniques. Cells were fixed w i t h 3.7% formaldehyde in PBS for 20 mins, and p e r m e a b i l i s e d with 0.1% Triton X-100 in PBS for 5 mins. To v i s u a l i s e actin, cells were incubated with TRITC-labelled p h a l l o i d i n as instructed by the m a n u f a c t u r e r s (Molecular Probes, Inc., Eugene, OR, USA). Immunofluorescence s t a i n i n g for e x t r a c e l l u l a r m a t r i x p r o t e i n s was c a r r i e d out as d e s c r i b e d p r e v i o u s l y (Warburton et al, 1982). Monospecific, polyclonal antisera to fibronectin, laminin and type IV collagen were produced and c h a r a c t e r i s e d (Warburton et al, 1981). Rabbit a n t i s e r u m to the fibronectin receptor p u r i f i e d from human placenta (integrin a551) was a gift from Dr R. Pytela (Pytela et al, 1985). FITC-labelled second antibodies were purchased from Nordic Immunological Laboratories, Maidenhead, UK. RESULTS D u r i n g the i n i t i a l p h a s e of c o n t a i n i n g m i c r o f i l a m e n t s form
cell s p r e a d i n g , actina p e r i p h e r a l ring close
Cell Biology International Reports, VoL 16, No. 3, 1992
Figure i. D i s t r i b u t i o n of fibronectin (a) and actin in Rama 25 cells 4h after a t t a c h m e n t . Fibronectin deposited beneath the peripheral, dorsal ring microfilaments. Bar 10~m.
209
(b) is of
to the dorsal surface of the cell. As cell s p r e a d i n g increases, the p e r i p h e r a l ring is d i s p l a c e d towards the p e r i n u c l e a r reglon. Fibronectin, but not l a m i n i n or type IV collagen, is d e p o s i t e d in p u n c t a t e s t r u c t u r e s which co-localise w i t h the p e r i p h e r a l microfilament b u n d l e s (Fig. 1 and 2). The fibronectin is p r o b a b l y of c e l l u l a r o r i g i n r a t h e r than s e r u m d e r i v e d b e c a u s e the same p a t t e r n s of f i b r o n e c t i n d e p o s i t i o n are o b s e r v e d in the a b s e n c e of serum. The d i s t r i b u t i o n of laminin is suggestive of a perinuclear, intracellular localisation. The p e r i p h e r a l ring becomes less condensed and develops into a series of c o n c e n t r i c circular filaments (Fig.2) . At this stage of cell spreading, the cells did not stain w i t h an a n t i b o d y to the a551, f i b r o n e c t i n receptor. Initially, the r e m a i n d e r of the c y t o p l a s m is l a r g e l y d e v o i d of m i c r o f i l a m e n t s , a l t h o u g h a few cells (~5%) d e v e l o p e x t e n s i v e stress fibres (not shown). Globular structures (geodomes) become apparent in the p e r i n u c l e a r r e g i o n and a s y s t e m of radial f i l a m e n t s e x t e n d s from these s t r u c t u r e s to the cell surface. At this stage, the e x t r a c e l l u l a r deposits of f i b r o n e c t i n were lost (Fig.3). As the cells form small colonies, the radial filaments disappear to be replaced by peripheral microfilament bundles. From this stage onwards, d e p o s i t i o n of fibronectin, laminin and type IV collagen takes place. These p r o t e i n s are l o c a t e d in p u n c t a t e structures b e n e a t h the basal surface of the cells and do not show any apparent co-distribution with the
210
Cell Biology International Reports, VoL 16, No. 3, 1992
14
` --
.'t: "
'
"S.~
Figure 2. Distribution of fibronectin (a), laminin ~c) and actin (b,d) in R~m@ 25 cells Sh after ~ttachment. The cells begin to extend lamellae and the p e r i p h e r a l microfilament ring becomes less condensed. Fibronectin, but not laminin, is d e p o s i t e d b e n e a t h the remnants of the p e r i p h e r a l ring. Laminin is r e s t r i c t e d to the perinuclear region. Bar 10~m.
peripheral microfilament bundles (Fig.4). Cells at the p e r i p h e r y of colonies extend lamellae. The p e r i p h e r a l ring of filaments forms polygonal structures from which stress fibres extend into the lamellae. Both fibronectin and its receptor (a551-integrin) c o - l o c a l i s e with the ends of the stress fibres in the lamellae (Fig.5).
Cell Biolouv International Reports, Vol. 16, No. 3, 1992
211
Figure 3. Distribution of fibronectin (a) and a¢tin (b) in R a m a 25 cells 10h after a t t a c h m e n t . Many cells develop an e x t e n s i v e s y s t e m of g e o d o m e s from w h i c h a c t i n - c o n t a i n i n g filaments radiate to the cell surface. Bar 20~m. DISCUSSION
The a c t i n - c o n t a i n i n g microfilament system of Rama 25 cells is a highly dynamic structure during cell attachment and spreading. The peripheral m i c r o f i l a m e n t bundles o b s e r v e d at early stages of cell spreading, in Rama 25 and other epithelial cells (Herman et al, 1981), is d i s p l a c e d from the cell p e r i p h e r y as the c y t o p l a s m extends. During this period, fibronectin is d e p o s i t e d in p u n c t a t e structures beneath the actin filaments. As cell spreading increases, the peripheral ring is r e p l a c e d by stress fibres that radiate from p o l y g o n a l structures in the perinuclear region to the cell surface. These polygonal structures or "geodomes" have been d e s c r i b e d in both epithelial and m e s e n c h y m a l cells (Ireland and Voon 1981; Mochizuki et al, 1988). It has been suggested that they p l a y a role in o r g a n i s i n g microfilaments into stress fibres (Gordon and Bushnell 1979). This v i e w is s u p p o r t e d by our f i n d i n g that polygonal structures also appear in Rama 25 cells in the p e r i p h e r a l ring of filaments when stress fibres extend into lamellae. The peripheral ring of actin filaments is p r o b a b l y analagous to the apical m i c r o f i l a m e n t ring o b s e r v e d in epithelia in vivo (Spooner 1975). In vivo, the a p i c a l ring is a n c h o r e d to j u n c t i o n a l complexes rather than receptors for extracellular matrix proteins
21 2
Cell Biology International Reports, VoL 16, No. 3, 1992
;i,:¸
F i g u r e 4. D i s t r i b u t i o n of f i b r o n e c t i n (a), l a m i n i n (c) ~ n d a c t i n (b,4) in c o l o n i e s of R a m a 2~ c e l l s 48h a f t e r attachment. There is n o a p p a r e n t co-distribution b e t w e e n e x t r a c e l l u l a r d e p o s i t s of f i b r o n e c t i n o r l a m i n i n and actin filament bundles. Bar 10~m.
Cell Biology International Reports, VoL 16, No. 3, 1992
213
'"
c
%tI
.
.
.
.
.
Figure 5. Distribution of fibron@ctin (a,c) , f i b r Q n e c t i n receptor {~) and actin (b) in the lamellae Qf R a m a 25 cells at th@ @dges of colonies. Actin filaments extend from polygonal arrays into the lamellae. There is c o - d i s t r i b u t i o n b e t w e e n actin and e x t r a c e l l u l a r d e p o s i t s of f i b r o n e c t i o n . Fibronectin r e c e p t o r is l o c a l i s e d at the ends of the f i b r o n e c t i n filaments. Bar 5~m.
Cell Biology Intemational Reports, VoL 16, No. 3, 1992
214
and this w o u l d e x p l a i n actin, f i b r o n e c t i n and cells.
the lack of c o - l o c a l i s a t i o n l a m i n i n in c o l o n i e s of R a m a
of 25
In cell a t t a c h m e n t assays, Rama 25 cells a t t a c h e q u a l l y to f i b r o n e c t i n and laminin ( u n p u b l i s h e d o b s e r v a t i o n s ) . The failure of these cells therefore to deposit d e t e c t a b l e amounts of laminin during cell a t t a c h m e n t and s p r e a d i n g may seem surprising. However, f i b r o n e c t i n has been r e p o r t e d to p r o m o t e the a t t a c h m e n t of several types of e p i t h e l i a l cell (Burrill et al, 1981; C l a r k et al, 1985). C e l l s a t t a c h to f i b r o n e c t i n by a n u m b e r of integrin receptors. The a 5 5 1 - i n t e g r i n is a s p e c i f i c r e c e p t o r for f i b r o n e c t i n and interacts with the RGD cell binding site of f i b r o n e c t i n (Pytela et al, 1985). A l t h o u g h a551 occurs on several cell types in vitro, it is less abundant in vivo (Albelda and Buck 1990). Other integrins, eg.a351, also function as fibronectin receptors. However, a351 also acts as a r e c e p t o r for laminin and collagens (Takada et al, 1988). Other integrins, eg. a651 and a654, also act as l a m i n i n receptors (Sonnenberg et al, 1990). In c u l t u r e d f i b r o b l a s t s , the f i b r o n e c t i n r e c e p t o r a c c u m u l a t e s in focal c o n t a c t s where it c o - l o c a l i s e s with the ends of stress fibres and f i b r o n e c t i n b o u n d to the s u b s t r a t u m (Chen et al, 1985). The receptor has been i m p l i c a t e d in cell a d h e s i o n but is not e s s e n t i a l for cell s p r e a d i n g or m i g r a t i o n (Akiyama et al, 1989). In Rama 25 cells, the a561 r e c e p t o r appears to be i n v o l v e d in the a d h e s i o n of l a m e l l a e and the a n c h o r i n g of stress fibres. It does not appear to be i n v o l v e d in the initial stages of Rama 25 cell a d h e s i o n and spreading. The early c o - a l i g n m e n t of m i c r o f i l a m e n t s and f i b r o n e c t i n s u g g e s t s that o t h e r f i b r o n e c t i n receptors may be involved. Further analysis of e x t r a c e l l u l a r m a t r i x - c y t o s k e l e t o n interaction in n o r m a l and t r a n s f o r m e d e p i t h e l i a l cells m a y h e l p to e l u c i d a t e the c o n t r i b u t i o n of s u c h i n t e r a c t i o n s to f u n c t i o n a l a s p e c t s of e p i t h e l i a l cell b e h a v i o u r , eg. cell m o t i l i t y and differentiation.
ACKNOWLEDGEMENT
S
We t h a n k Dr Robert P y t e l a for the gift of a n t i s e r u m to the f i b r o n e c t i n receptor. This work was s u p p o r t e d by grants f r o m the Cancer Research Campaign, Medical R e s e a r c h Council and SmithKline Foundation.
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with
monoclonal
antibodies:
roles
in
cell
Cell Biology International Reports, Vol. 16, No. 3, 1992 adhesion, migration, m a t r i x a s s e m b l y and organization. J. Cell Biol. 109, 863-875.
21 5 cytoskeletal
Albelda, S.M. and Buck, C.A. (1990).Integrins and other cell adhesion molecules. FASEB J 4, 2868-2880. Barcellos-Hoff, M.H., Aggeler, J., Ram, T.G. and Bissell, M.J. (1989). F u n c t i o n a l differentiation and a l v e o l a r m o r p h o g e n e s i s of p r i m a r y m a m m a r y c u l t u r e s on r e c o n s t i t u t e d b a s e m e n t membrane. D e v e l o p m e n t 105, 223235. Ben-Ze'ev, A. cells. Biochim.
(1985). Biophys.
The Acta
cytoskeleton 780, 197-212.
Bennett, D.C., Peachey, L.A., Rudland, P.S. (1978). A possible line. Cell 15, 283-298.
in
cancer
Durbin, H. mammary stem
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Burrill, P.H., Bernardini, I., Kleinman, H.K. and Kretchmer, N. (1981). Effect of serum, fibronectin and laminin on adhesion of rabbit intestinal e p i t h e l i a l cells in culture..J. Supramolec. Struct. Cell. Biochem. 16, 3 8 5 - 3 9 2 . Chen,W.-T., Hasegawa, E., Hasegawa, T., Weinstock, C. and Yamada, K.M. (1985). D e v e l o p m e n t of cell surface linkage complexes in c u l t u r e d fibroblasts. J. Cell Biol. 100, 1 1 0 3 - 1 1 1 4 . Clark, R.A.F., Folkvord, J.M. and Wertz, R.L. (1985). F i b r o n e c t i n , as well as o t h e r e x t r a c e l l u l a r m a t r i x proteins, m e d i a t e human k e r a t i n o c y t e adherence. J. Invest. Dermatol. 84, 378-383. Glenney, J.R. and Zokas, L. (1989). N o v e l tyrosine k i n a s e s u b s t r a t e s from Rous s a r c o m a v i r u s - t r a n s f o r m e d cells are p r e s e n t in the m e m b r a n e skeleton. J. Cell Biol. 108, 2 4 0 1 - 2 4 0 8 . Gordon, W.E. and Bushnell, A. (1979). Immunofluorescent and u l t r a s t r u c t u r a l s t u d i e s of p o l y g o n a l m i c r o f i l a m e n t networks in r e s p r e a d i n g n o n - m u s c l e cells. Exp. Cell Res. 120, 335-348. Herman, I.M., Crisona, N.J. and Pollard, T.D. (1981). R e l a t i o n b e t w e e n cell activity and the distribution of cytoplasmic actin and myosin. J. Cell Biol. 90, 84-91. Ireland, G.W. and Voon, F.C.T. n e t w o r k s in living chick e m b r y o n i c 52, 55-69.
(1981) . P o l y g o n a l cells. J. Cell. Sci.
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Mochizuki, Y., Furukawa, K., Mitaka, T., Yokoi, T. and Kodama, T. (1988). Polygonal networks, "geodomes", of adult rat hepatocytes in p r i m a r y culture. Cell Biol. Int. Rep. 12, 1-7. Pytela, R., Pierschbacher, M.D. and Ruoslahti, E. (1985). I d e n t i f i c a t i o n and i s o l a t i o n of a 140kd cell surface glycoprotein with properties expected of a f i b r o n e c t i n receptor. Cell 40, 191-198. Seely, K.A. and Aggeler, J. (1991). Modulation of milk protein synthesis through alteration of the c y t o s k e l e t o n in mouse m a m m a r y e p i t h e l i a l cells c u l t u r e d on a r e c o n s t i t u t e d basement membrane. J. Cell. Physiol. 146, 117-130.
Sonnenberg, A., Linders, C.J.T., Daams, J.H. and Kennel, S.J. (1990). The ~ 6 ~ i (VLA-6) and ~6~4 p r o t e i n complexes: tissue d i s t r i b u t i o n and b i o c h e m i c a l properties. J. Cell. Sci. 96, 207-217. Spooner, B.S. (1975). Microfilaments, microtubules and e x t r a c e l l u l a r materials in morphogenesis. B i o S c i e n c e 25, 440-450. Takada, Y. , Wayner, E.A. , Carter, W.G. and Hemler, M.E. (1988) . E x t r a c e l l u l a r matrix receptors, ECMRII and ECMRJ, for collagen and fibronectin c o r r e s p o n d to VLA-2 and VLA-3 in the V L A f a m i l y of heterodimers. J. Cell Biol. 37, 385-393. Warburton, M.J., Ferns, S.A. and Rudland, P.S. (1982). Enhanced synthesis of basement membrane proteins d u r i n g the d i f f e r e n t i a t i o n of rat m a m m a r y tumour epithelial cells into m y o e p i t h e l i a l - l i k e cells in vitro. Exp. Cell Res. 137,'373-380. Warburton, M.J. , Ormerod, E.J. , Monaghan, P. , Ferns, S.A. and Rudland, P.S. (1981) . C h a r a c t e r i z ation of a • m y o e p i t h e l i a l cell line d e r i v e d from a neonatal rat mammary gland. J. Cell Biol. 91, 827-836. Watanabe, H., Carmi, P., Hogan, V., Raz, T., Silletti, S., Nabi, I.R. and Raz, A. (1991). P u r i f i c a t i o n of human t u m o r cell m o t i l i t y f a c t o r and m o l e c u l e c l o n i n g of its receptor. J. Biol. Chem. 266, 13442-13448. Wicha, M.S., Lowrie, G., Kohn, E., Bagavandoss, P. and Mahn, T. (1982). E x t r a c e l l u l a r m a t r i x p r o m o t e s m a m m a r y e p i t h e l i a l growth and d i f f e r e n t i a t i o n in vitro. Proc. Natl. Acad. Sci. USA. 79, 3213-3217. Paper received 14.09.91. Revised paper accepted 21.01.92.
OF
FIBRONECTIN
CYTOSKELETON
Sharon
A.
DURING
Ferns, and
Department
IN THE
THE
ORGANISATION
SPREADING
EPITHELIAL
CELLS
Rosemary
Kimbell,
Michael
J.
of Histopathology,
Medical School,
207
OF
OF
RAT
Jane
A.
THE
MAMMARY
Aitken
Warburton.
St George's
London SWI7 ORE,
Hospital
UK.
ABSTRACT
The c o r r e l a t i o n between the e x t r a c e l l u l a r d e p o s i t i o n of f i b r o n e c t i n and the d e v e l o p m e n t of the a c t i n - c o n t a i n i n g cytoskeleton was studied during the a t t a c h m e n t and s p r e a d i n g of the rat m a m m a r y e p i t h e l i a l cell line Rama 25. D u r i n g the initial phase of cell spreading, actin is l o c a l i s e d in p e r i p h e r a l m i c r o f i l a m e n t bundles. As cell spreading .increases, the peripheral ring is d i s p l a c e d towards the p e r i n u c l e a r region. Fibronectin, d e p o s i t e d b e n e a t h the basal surface, c o - l o c a l i s e s with the a c t i n - c o n t a i n i n g p e r i p h e r a l ring. The p e r i p h e r a l ring s u b s e q u e n t l y disappears and is replaced by a system of radial microfilaments that extend from the perinuclear r e g i o n to the cell p e r i p h e r y . At this stage, there is no correlation b e t w e e n the d i s t r i b u t i o n of f i b r o n e c t i n and actin. As cells f o r m colonies, radial m i c r o f i l a m e n t bundles are r e p l a c e d by p e r i p h e r a l microfilament b u n d l e s w h i c h do not c o - l o c a l i s e with fibronectin. Cells at the edges of c o l o n i e s e x t e n d lamellae that contain m i c r o f i l a m e n t stress fibres. In these structures t h e r e is c o - l o c a l i s a t i o n of actin, fibronectin and the a551-integrin fibronectin receptor. INTRODUCTION
The functional d i f f e r e n t i a t i o n of mammary cells in v i t r o is dependent on the composition of the s u b s t r a t u m (Wicha et al, 1982). The a b i l i t y of m a m m a r y e p i t h e l i a l cells to s y n t h e s i s e milk p r o t e i n s in r e s p o n s e to l a c t o g e n i c hormones is m a x i m i s e d by g r o w t h on s u b s t r a t a (eg. r e c o n s t i t u t e d b a s e m e n t m e m b r a n e gels) that a l l o w cells to p o l a r i s e and form duct-like structure (Barcellos-Hoff et al, 1989). The e x t r a c e l l u l a r m a t r i x influences cell behaviour partly through binding to cell surface r e c e p t o r s whose c y t o p l a s m i c d o m a i n s interact with the c y t o s k e l e t o n (Albelda and Buck 1990). Evidence has been
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p r e s e n t e d that the i n t e g r i t y of the c y t o s k e l e t o n is r e q u i r e d for the h o r m o n a l i n d u c t i o n of m i l k p r o t e i n synthesis (Seely and A g g e l e r 1991). The c y t o s k e l e t o n also p a r t l y d e t e r m i n e s cell shape, g r o w t h c o n t r o l and the e x p r e s s i o n of the m a l i g n a n t p h e n o t y p e (Ben-Ze'ev 1985). D u r i n g transformation, changes in the d i s t r i b u t i o n of e x t r a c e l l u l a r matrix components and t h e i r r e c e p t o r s are a c c o m p a n i e d by a r e a r r a n g e m e n t of the cytoskeleton. The structure of the c y t o s k e l e t o n may be further m o d i f i e d by t y r o s i n e kinases for w h i c h several c y t o s k e l e t a l substrates exist (Glenney and Zokas 1989). Interactions of tumour cells with c h e m o t a c t i c or m o t i l ity factors (Watanabe et al, 1991) p r e s u m a b l y i n v o l v e the m i c r o f i l a m e n t system. Whereas considerable progress has been made in u n d e r s t a n d i n g microfilament and extracellular matrix interactions in normal and transformed fibroblasts, relatively little is k n o w n about t h e s e p r o c e s s e s in e p i t h e l i a l cells. We n o w report on the r e l a t i o n s h i p between e x t r a c e l l u l a r m a t r i x d e p o s i t i o n and m i c r o f i l a m e n t o r g a n i s a t i o n d u r i n g the a t t a c h m e n t and s p r e a d i n g of a rat m a m m a r y e p i t h e l i a l cell line. EXPERIMENTAL
PROCEDURES
Tissue Culture. Rama 25 cells were cultured in D u l b e c c o ' s m o d i f i e d Eagles m e d i u m s u p p l e m e n t e d with 5% fetal calf serum, 5 0 n g / m l h y d r o c o r t i s o n e and 5 0 n g / m l insulin. This cell line was isolated from a d i m e t h y l b e n z a n t h r a c e n e - i n d u c e d rat m a m m a r y t u m o u r (Bennett et al, 1978). Immunofluorescence Techniques. Cells were fixed w i t h 3.7% formaldehyde in PBS for 20 mins, and p e r m e a b i l i s e d with 0.1% Triton X-100 in PBS for 5 mins. To v i s u a l i s e actin, cells were incubated with TRITC-labelled p h a l l o i d i n as instructed by the m a n u f a c t u r e r s (Molecular Probes, Inc., Eugene, OR, USA). Immunofluorescence s t a i n i n g for e x t r a c e l l u l a r m a t r i x p r o t e i n s was c a r r i e d out as d e s c r i b e d p r e v i o u s l y (Warburton et al, 1982). Monospecific, polyclonal antisera to fibronectin, laminin and type IV collagen were produced and c h a r a c t e r i s e d (Warburton et al, 1981). Rabbit a n t i s e r u m to the fibronectin receptor p u r i f i e d from human placenta (integrin a551) was a gift from Dr R. Pytela (Pytela et al, 1985). FITC-labelled second antibodies were purchased from Nordic Immunological Laboratories, Maidenhead, UK. RESULTS D u r i n g the i n i t i a l p h a s e of c o n t a i n i n g m i c r o f i l a m e n t s form
cell s p r e a d i n g , actina p e r i p h e r a l ring close
Cell Biology International Reports, VoL 16, No. 3, 1992
Figure i. D i s t r i b u t i o n of fibronectin (a) and actin in Rama 25 cells 4h after a t t a c h m e n t . Fibronectin deposited beneath the peripheral, dorsal ring microfilaments. Bar 10~m.
209
(b) is of
to the dorsal surface of the cell. As cell s p r e a d i n g increases, the p e r i p h e r a l ring is d i s p l a c e d towards the p e r i n u c l e a r reglon. Fibronectin, but not l a m i n i n or type IV collagen, is d e p o s i t e d in p u n c t a t e s t r u c t u r e s which co-localise w i t h the p e r i p h e r a l microfilament b u n d l e s (Fig. 1 and 2). The fibronectin is p r o b a b l y of c e l l u l a r o r i g i n r a t h e r than s e r u m d e r i v e d b e c a u s e the same p a t t e r n s of f i b r o n e c t i n d e p o s i t i o n are o b s e r v e d in the a b s e n c e of serum. The d i s t r i b u t i o n of laminin is suggestive of a perinuclear, intracellular localisation. The p e r i p h e r a l ring becomes less condensed and develops into a series of c o n c e n t r i c circular filaments (Fig.2) . At this stage of cell spreading, the cells did not stain w i t h an a n t i b o d y to the a551, f i b r o n e c t i n receptor. Initially, the r e m a i n d e r of the c y t o p l a s m is l a r g e l y d e v o i d of m i c r o f i l a m e n t s , a l t h o u g h a few cells (~5%) d e v e l o p e x t e n s i v e stress fibres (not shown). Globular structures (geodomes) become apparent in the p e r i n u c l e a r r e g i o n and a s y s t e m of radial f i l a m e n t s e x t e n d s from these s t r u c t u r e s to the cell surface. At this stage, the e x t r a c e l l u l a r deposits of f i b r o n e c t i n were lost (Fig.3). As the cells form small colonies, the radial filaments disappear to be replaced by peripheral microfilament bundles. From this stage onwards, d e p o s i t i o n of fibronectin, laminin and type IV collagen takes place. These p r o t e i n s are l o c a t e d in p u n c t a t e structures b e n e a t h the basal surface of the cells and do not show any apparent co-distribution with the
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14
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'
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Figure 2. Distribution of fibronectin (a), laminin ~c) and actin (b,d) in R~m@ 25 cells Sh after ~ttachment. The cells begin to extend lamellae and the p e r i p h e r a l microfilament ring becomes less condensed. Fibronectin, but not laminin, is d e p o s i t e d b e n e a t h the remnants of the p e r i p h e r a l ring. Laminin is r e s t r i c t e d to the perinuclear region. Bar 10~m.
peripheral microfilament bundles (Fig.4). Cells at the p e r i p h e r y of colonies extend lamellae. The p e r i p h e r a l ring of filaments forms polygonal structures from which stress fibres extend into the lamellae. Both fibronectin and its receptor (a551-integrin) c o - l o c a l i s e with the ends of the stress fibres in the lamellae (Fig.5).
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Figure 3. Distribution of fibronectin (a) and a¢tin (b) in R a m a 25 cells 10h after a t t a c h m e n t . Many cells develop an e x t e n s i v e s y s t e m of g e o d o m e s from w h i c h a c t i n - c o n t a i n i n g filaments radiate to the cell surface. Bar 20~m. DISCUSSION
The a c t i n - c o n t a i n i n g microfilament system of Rama 25 cells is a highly dynamic structure during cell attachment and spreading. The peripheral m i c r o f i l a m e n t bundles o b s e r v e d at early stages of cell spreading, in Rama 25 and other epithelial cells (Herman et al, 1981), is d i s p l a c e d from the cell p e r i p h e r y as the c y t o p l a s m extends. During this period, fibronectin is d e p o s i t e d in p u n c t a t e structures beneath the actin filaments. As cell spreading increases, the peripheral ring is r e p l a c e d by stress fibres that radiate from p o l y g o n a l structures in the perinuclear region to the cell surface. These polygonal structures or "geodomes" have been d e s c r i b e d in both epithelial and m e s e n c h y m a l cells (Ireland and Voon 1981; Mochizuki et al, 1988). It has been suggested that they p l a y a role in o r g a n i s i n g microfilaments into stress fibres (Gordon and Bushnell 1979). This v i e w is s u p p o r t e d by our f i n d i n g that polygonal structures also appear in Rama 25 cells in the p e r i p h e r a l ring of filaments when stress fibres extend into lamellae. The peripheral ring of actin filaments is p r o b a b l y analagous to the apical m i c r o f i l a m e n t ring o b s e r v e d in epithelia in vivo (Spooner 1975). In vivo, the a p i c a l ring is a n c h o r e d to j u n c t i o n a l complexes rather than receptors for extracellular matrix proteins
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;i,:¸
F i g u r e 4. D i s t r i b u t i o n of f i b r o n e c t i n (a), l a m i n i n (c) ~ n d a c t i n (b,4) in c o l o n i e s of R a m a 2~ c e l l s 48h a f t e r attachment. There is n o a p p a r e n t co-distribution b e t w e e n e x t r a c e l l u l a r d e p o s i t s of f i b r o n e c t i n o r l a m i n i n and actin filament bundles. Bar 10~m.
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c
%tI
.
.
.
.
.
Figure 5. Distribution of fibron@ctin (a,c) , f i b r Q n e c t i n receptor {~) and actin (b) in the lamellae Qf R a m a 25 cells at th@ @dges of colonies. Actin filaments extend from polygonal arrays into the lamellae. There is c o - d i s t r i b u t i o n b e t w e e n actin and e x t r a c e l l u l a r d e p o s i t s of f i b r o n e c t i o n . Fibronectin r e c e p t o r is l o c a l i s e d at the ends of the f i b r o n e c t i n filaments. Bar 5~m.
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and this w o u l d e x p l a i n actin, f i b r o n e c t i n and cells.
the lack of c o - l o c a l i s a t i o n l a m i n i n in c o l o n i e s of R a m a
of 25
In cell a t t a c h m e n t assays, Rama 25 cells a t t a c h e q u a l l y to f i b r o n e c t i n and laminin ( u n p u b l i s h e d o b s e r v a t i o n s ) . The failure of these cells therefore to deposit d e t e c t a b l e amounts of laminin during cell a t t a c h m e n t and s p r e a d i n g may seem surprising. However, f i b r o n e c t i n has been r e p o r t e d to p r o m o t e the a t t a c h m e n t of several types of e p i t h e l i a l cell (Burrill et al, 1981; C l a r k et al, 1985). C e l l s a t t a c h to f i b r o n e c t i n by a n u m b e r of integrin receptors. The a 5 5 1 - i n t e g r i n is a s p e c i f i c r e c e p t o r for f i b r o n e c t i n and interacts with the RGD cell binding site of f i b r o n e c t i n (Pytela et al, 1985). A l t h o u g h a551 occurs on several cell types in vitro, it is less abundant in vivo (Albelda and Buck 1990). Other integrins, eg.a351, also function as fibronectin receptors. However, a351 also acts as a r e c e p t o r for laminin and collagens (Takada et al, 1988). Other integrins, eg. a651 and a654, also act as l a m i n i n receptors (Sonnenberg et al, 1990). In c u l t u r e d f i b r o b l a s t s , the f i b r o n e c t i n r e c e p t o r a c c u m u l a t e s in focal c o n t a c t s where it c o - l o c a l i s e s with the ends of stress fibres and f i b r o n e c t i n b o u n d to the s u b s t r a t u m (Chen et al, 1985). The receptor has been i m p l i c a t e d in cell a d h e s i o n but is not e s s e n t i a l for cell s p r e a d i n g or m i g r a t i o n (Akiyama et al, 1989). In Rama 25 cells, the a561 r e c e p t o r appears to be i n v o l v e d in the a d h e s i o n of l a m e l l a e and the a n c h o r i n g of stress fibres. It does not appear to be i n v o l v e d in the initial stages of Rama 25 cell a d h e s i o n and spreading. The early c o - a l i g n m e n t of m i c r o f i l a m e n t s and f i b r o n e c t i n s u g g e s t s that o t h e r f i b r o n e c t i n receptors may be involved. Further analysis of e x t r a c e l l u l a r m a t r i x - c y t o s k e l e t o n interaction in n o r m a l and t r a n s f o r m e d e p i t h e l i a l cells m a y h e l p to e l u c i d a t e the c o n t r i b u t i o n of s u c h i n t e r a c t i o n s to f u n c t i o n a l a s p e c t s of e p i t h e l i a l cell b e h a v i o u r , eg. cell m o t i l i t y and differentiation.
ACKNOWLEDGEMENT
S
We t h a n k Dr Robert P y t e l a for the gift of a n t i s e r u m to the f i b r o n e c t i n receptor. This work was s u p p o r t e d by grants f r o m the Cancer Research Campaign, Medical R e s e a r c h Council and SmithKline Foundation.
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