Fibronectin
Fibronectin (FN) is a very large (Mr=500,000) dim eric glycoprotein com posed of tw o sim ilar subunits (each alm ost 2500 am ino acid residues long) linked by a pair of disulphide bonds
aro u n d half of all m em brane glycoproteins. It is an extrem ely effective b ro ad range natural
adhesive factor (Ruoslahti, 1988; Yamada, 1989; Aota et al, 1991) and prom otes the attachm ent
and spreading of m any cell types, including BHK and CHO cells (Bames & Sato, 1980), neurons
(Me Guire & Seeds, 1990), fibroblasts (Tremble et al, 1993) and smooth m uscle cells (Di Milla et
al, 1993). A com ponent of both plasm a and cell secreted m atrix (Vaheri & M osher, 1978; Hynes
& Yam ada, 1982), FN functions as a cellular adhesion factor by binding to COL, heparin and
other cell glycosam inoglycans (Aota et al, 1991). The assem bly of FN into the ECM has been
review ed (Mosher, 1993). FN has been added to serum -free culture system s to im prove cell adhesion and grow th and m ore recently show n to be useful in the grow th of norm al lung and
cystic fibrosis epithelial cells in low serum culture (Bames & Sato, 1980; Schwiebert et al, 1992).
It m ay be used either as a thin coating for tissue culture plastic or as a soluble m edia additive.
In short or long-term culture it has been dem onstrated that FN expression is lost on
transform ed glial cells to some degree (Bigner et al, 1981a; Sherbet et al, 1982). It has been
suggested that the absence of surface FN m ay account for the inability of hum an glioma cells to interact a n d attach to the basem ent m em brane of capillaries, hence explaining the lack of
system ic m étastasés in these tum ours (Sherbet et al, 1987). This subject w ill be discussed in
m ore detail in the imm unocytochemistry chapter.
Lam inin
Lam inin (LMN) is a large (Mr=900,000) glycoprotein and is the m ajor non-collagenous glycoprotein com ponent of BMs and is com posed of three polypeptide chains w ith a m ulti
dom ain structure (Beck et al, 1990). It belongs to a family of proteins w ith several genetically
distinct subunit chains that are present in num erous isoforms (Tryggvason, 1993). The structure and prop erties of LMN have been w idely described (M artin & Timpl, 1987; Engel, 1992; T ryggvason, 1993). It interacts w ith o ther BM com ponents, in clu d in g type IV COL,
proteoglycan, entactin, heparin sulphate and related polysaccharides (Timpl et al, 1983; Engel,
1992). LM N has been show n to have a num ber of biological activities in vitro, including the
ability to influence cell adhesion, growth, morphology, differentiation, m atrix assembly and cell
m igration (Kleinman ef al, 1985; Beck et al 1990) of a variety of cells. It prom otes the attachm ent
of various epithelial cells to plastic or to type IV COL-coated surfaces (Terranova et al, 1980;
V lodavsky & Gospodarowicz, 1981). LMN m ay regulate the grow th of a variety of cell types,
including m acrophages (Me Kay et al, 1992), kératinocytes (Adams & W att, 1991), m elanom a
cells (Yam am ura et al, 1993), m s-transform ed cells (Chambers et al, 1993) and m ouse em bryo
stem cells (Cooper et al, 1991). LMN is also thought to be im portant in developm ent and has
been identified in the 4-cell stage embryo (Cooper & Mac Queen, 1983).
LMN has been reported to be involved in the grow th of neurons (Kleinman ef al, 1988),
in the grow th cone elongation of sensory neurons (Lamoureux et al, 1992) as well as influencing
reported th at radioactively labelled LM N bin d s specifically to the surface of NG108-15 neuroblastom a/gliom a cells via three m em brane proteins of 110 kDa, 180 kDa and 67 kDa. The 110 kDa protein occurs in a variety of epithelial cells and in brain, w hereas the 180 kDa protein is neuron specific. Antibodies against the 110 kDa and 180 kDa proteins inhibit LM N-induced neurite outgrow th, w hilst antibodies directed against the 67 kDa protein do not.
Unlike FN, LMN is able to bind either to the cell surface or to COL, although it appears to be m ore effective as an attachm ent protein for epithelial cells w hen b ound to type IV COL. Certain fibroblasts do not recognise LMN and will not survive in culture if excess lam inin is
present (Terranova et al, 1983).
As w ell as adhesive properties other changes observed include changes in cell shape
and have been related to changes in cell grow th (FoUonan & Moscona, 1978; G ospodarowicz et
al, 1978). Sugrue and H ay (1981), observed that LMN altered cellular m orphology and that
epithelial cells w ith o u t a BM form ed blebs on th eir surface indicating th at the basal plasm alem m a was unstable. Observations from other studies include elongation of Schwann cells (Palm & Furcht, 1983) and spreading of HT-1080 cells (Gold & Pearlstein, 1980). LMN therefore possibly stim ulates a morphological feature of these cells, which is m ore characteristic
of the in vivo m orphology for these particular cell populations.
Tenascin
Tenascin (TN) also know n as cytotactin is another ECM glycoprotein but its tissue distribution
is m ore lim ited than th at of the other ECM glycoproteins (C hiquet-Ehrism ann et al, 1986;
Erickson & Bourdon, 1989). TN is transiently present in the dense m esenchym e surrounding
several developing organs, i.e., m am m ary gland during embryogenesis (Chiquet-Ehrismann et
al, 1986), kidney (A ufderheide et al, 1987) and in both CNS and PNS (G rum et et al, 1985;
Bronner-Fraser, 1988) w here TN expression is thought to be related to cell proliferation and m igration as well as rem odelling of the ECM (Bronner-Fraser, 1988; Erickson & Bourdon, 1989).
Tenascin appears to be p art of a family consisting of three related proteins, tenascin-C (TN-C), tenascin-R (TN-R) and tenascin-X (TN-X), properties of w hich have been recently discussed (Erickson, 1993). TN-C is the first m em ber of the TN fam ily to have been characterised and is expressed in developing brain, cartilage and m esenchym e and is also re expressed in tum ours, w ound healing and inflamm ation (Erickson, 1993). TN-R was originally
described u n d er the nam es restrictin in chicken (Rathjen et al, 1991) and as Jl-160/180 in rat
(Pesheva et al, 1989). TN-X w as originally reported as a partial sequence encoded by gene X
(Morel et al, 1989) and recently alm ost aU of the sequence of this large protein has been reported
(Bristow et al, 1993). TN-X mRNA is m ainly expressed in foetal muscle (smooth m uscle of gut,
skeletal muscle and heart) and testis w ith a lower level in foetal adrenal gland, kidney and lung (Bristow et al, 1993).
Studies have show n that m ore than half of the TN m olecule is hom ologous to the
amino acid sequence of FN (Jones et al, 1988; Erickson & Bourdon, 1989), It seems that these two
glycoproteins have opposing functions, FN has an inhibitory effect on chondrogenic and
osteogenic differentiation in vitro and in vivo, w hilst TN prom otes such differentiation (Mackie
et al, 1987). The attachm ent and spreading of cells on FN are inhibited by soluble TN (Bourdon
& Ruoslahti, 1989; Lotz et al, 1989). TN was show n to inhibit neurite outgrow th from dorsal root
ganglia on FN and LMN substrates (Crossin et al, 1990) w hilst it w as able to enhance neurite
grow th w hen neurons w ere cultured on PLL (Lochter et al, 1991). Recently, w hen cells were
offered a mixed substrata w ith other ECM proteins, tw o TN-R isoforms and TN-C derived from m ouse b rain selectively inhibited FN -dependent cell adhesion and n eu rite outgrow th and
affected cell m orphology of different m esenchymal and neural cells (Pesheva et al, 1994).
V itronectin
V itronectin (VT) is a glycoprotein present in h u m an plasm a and serum an d is capable of m ediating attachm ent, m igration, proliferation and spreading of a n u m b er of cell types,
including fibroblastic and epithelial cells (Bames & Silnutzer, 1983; H aym an et al, 1985) as well
as prom oting retinal neurite outgrow th (Neugebauer et al, 1991) and for studying cell m igration
(Leavesley et al, 1992). It is commonly referred to as the "serum spreading factor". D uring cell
spreading, the attached cell flattens or spreads, usually assum ing the shape characteristic of its differentiated state. This is induced by the form ation of focal adhesions or sites of strongest adhesion at the cell surface that m ake contact w ith the substratum (Harris, 1973) d ue to cell attachment.
Entactin/nidogen
Entactin and nidogen are identical sulphated glycoproteins originally identified in cultured cells, as in m ouse endoderm al cell line and found in the surface of epithelial cells of tubules and
glom eruli adjacent to BMs (Carlin et al, 1981) b ut subsequently also isolated from m ouse EHS
tu m our (Timpl et al, 1983). Entactin has a M r of 150,000 and w as isolated from em bryonic
neurons from chick dorsal root ganglia (Carbonetto et al, 1983). N idogen has been show n to
have the potential to bind to other ECM com ponents and is thought to be a m ajor m ediator in
BM assem bly (Aum ailley et al, 1993). The LM N-nidogen complex binds the core protein of
heparin-sulphate proteoglycan (Battaglia et al, 1992). This binding occurs via different globular
dom ains of nidogen (R eindardt et al, 1993) and is considered to be an essential step in
connecting netw orks of LMN and COL IV w ith BMs (Aumailley et al, 1993).
G lycosam inoglycans and proteoglycans
The glycosam inoglycans (GAGs) include four m ain groups divided according to their sugar residues, the type of linkage betw een these residues and the num ber and location of sulphate groups and include: (i) hyaluronic acid, (ii) chondroitin sulphate and derm atan sulphate, (iii)
heparin sulphate and heparin and (iv) keratin sulphate. These molecules are long unbranched polysaccharide chains composed of repeating disaccharide units. One of the tw o sugar residues in the repeating disaccharide is always an amino sugar. They play an im portant role in cellular attachm ent, provide cellular support and regulate cell biosynthesis and additionally have a role
in cell proliferation and differentiation (Ehas et al, 1988).
Proteoglycans are very large ECM m acromolecules that contain 90-95% carbohydrate by w eight in the form of m any long unbranched GAG side chains attached to a core protein m olecule (for review of structure see Kjellèn & Lindahl, 1991). These m olecules have been show n to be im portant in cell grow th and adhesion, receptor binding a n d transform ation
(Margolis et al, 1975; lozzo, 1984; Turley, 1984). Purified proteoglycans have been used to study
their interaction w ith soluble com ponents of cell culture m edia (lida et al, 1992) and grow th
factors, such as FGF, EGF and TGF-P (Klagsbrun & Baird, 1991; Ruoslahti & Yamaguchi, 1991).