The Fimbrin Family Of Actinbinding Proteins

Molecular Features of Fimbrin

Fimbrins are EF-hand calcium-binding proteins that actively participate in binding to and bundling of actin. In actin filaments, one molecule of fimbrin might bind eight actin monomers under optimal conditions (Namba et al. 1992). Fimbrins are highly conserved in evolution, with regard to both their structure and function. Fimbrin from diverse origins, e.g., from Dictyostelium discoideum to humans, share structural and biochemical properties. Fimbrin-like proteins have also been isolated from plants, such as wheat (Triticum aestivum) and Arabidopsis thaliana (Cruz-Ortega et al. 1997). The Dictyostelium fimbrin is a 67-kDa protein containing two EF-hands that are followed by two actin-binding sites (Prassler et al. 1997). The N-terminal actin-binding domain is a highly conserved domain that fimbrin shares with other actin-binding proteins. This N-terminal domain contains two tandem calponin homology (CH) domains which are implicated in the binding of F-actin (Goldsmith et al. 1997). Fimbrin-mediated cross-linking and bundling of actin is regulated by free calcium, which it binds with high affinity and selectivity. However, the optimal concentration for cross-linking of actin was determined to be below 0.15 ||M. This process was progressively inhibited at higher free calcium concentrations and halfmaximal inhibition of cross-linking occurred at 1.6 |M Ca2+ concentration (Pacaud and Derancourt, 1993). Pacaud and Derancourt (1993) also noted that calcium-binding of fimbrin, in the concentration range of 0.15 to 1.5 |M, produced confor-mational changes in the molecule, to which they attribute the calcium-mediated regulation of fimbrin function. It may be that the conformational alterations actuated by calcium at different concentrations could render actin cross-linking reversible. A further possibility has arisen from the studies of Hanein et al. (1997a) that fimbrin might induce conformational changes in actin itself. Both types of change will inevitably contribute to cell behaviour.

Fimbrins form a large family of proteins. Four genes encode fimbrin in Salmonella (Collinson et al. 1996). Three isoforms of fimbrin have been identified and designated as I-fimbrin, L-fimbrin, and T-fimbrin. I- and L-fimbrin are characteristically associated with intestinal and kidney epithelia, leukocytes, and tumours, whereas T-fimbrin shows a more general distribution in a variety of cells and tissues (Chafel, 1995).

Function of Fimbrin in Cytoskeletal Organisation

A major function of fimbrin appears to be in the assembly of actin filaments. Fimbrin as well as actin capping protein (CP) are required for proper assembly of these filaments in the yeast Saccharomyces cerevisiae. There is a reduced filament assembly and fimbrin in the CP mutants of the yeast. Actin obtained from CP mutants shows defects in polymerisation as well as in its binding to fimbrin (Karpova et al. 1995). In mammalian cells, Rho GTPases may mediate actin filament assembly and bundling. A constitutive expression of the Rho GTPase Cdc42Hs causes impairment of cytokinesis of HeLa cells. This seems to be a consequence of a reorganisation of F-actin with which, among other actin-binding proteins, T-fimbrin is associated (Dutartre et al. 1996). Functional conservation of fimbrin isoforms has been amply demonstrated by Adams et al. (1995), who found that human T- and L-fimbrin can substitute for the yeast fimbrin called Sac6p. T- and L-fimbrin were both able to complement the temperature-sensitive growth defect that is seen in sac6 null mutants, and they could also restore normal cytoskeletal organisation and cell shape in these mutants. The null mutants show defective sporulation, which is restored by human T- and L-fimbrin (Adams et al. 1995).

Fimbrin isoforms show not only a tissue-specific distribution, but the specificity appears also to extend to differentiation and morphogenesis. Fimbrin shows a definable temporal and spatial pattern of expression in the course of the development of the cochlea and may be involved in the formation of the inner and outer stereocilia of the hair cell (Zine et al. 1995). The differentiation of intestinal epithelium is associated with the expression of T-fimbrin in the apical area of the cell and L-fimbrin in the basal area, until 14.5 days of development. Both isoforms are said to be totally down-regulated in expression by day 16.5, but instead, I-fimbrin appears on day 14.5 of development to give the epithelium a brush border-like localisation. These findings, reported by Chafel et al. (1995), suggest possible differences in their function in the course of differentiation. A number of the events occurring in morphogenesis and differentiation require changing patterns of cell adhesion, for which fimbrin seems to be ideally placed. Babb et al. (1997) studied the localisation of fimbrin in mature as well as differentiating osteoclasts. Fimbrin is a component of the osteoclast adhesion complexes called podosomes. During migration podo-somes are found at the cell periphery. Microfilament organisation and podosome assembly is important in the rapid modulation of adhesion to the substratum, and in the motility of cells. T- and L-fimbrin, but not I-fimbrin occurred as an integral component of the core of the podosomes throughout the process of monocyte-derived osteoclast differentiation. The levels of T-fimbrin increased with and appeared to be related to the formation of podosomes.

The involvement of fimbrin in podosomes is compatible with the association of fimbrin with the adhesive organelles called fimbriae that have been described in bacteria. Fimbriae are fibre-like structures that mediate the attachment of bacteria to host cells. These are assembled from fimbrin subunits (Smyth et al. 1996). Frederick et al. (1996) investigated the adhesive interactions between lymphokine-activated killer (LAK) cells and came to the conclusion that fimbrin might be an important factor in intercellular adhesive contacts. They also showed that contact between LAK cells and target tumour cells, namely SK-Mel-1 human melanoma cells and Raji lymphoma cells, leads to the phosphorylation of L-fimbrin of the LAK cells. S.L. Jones et al. (1998) have also suggested that induction of fimbrin phos-phorylation is an important step in fimbrin function. However, as discussed below, the question of whether phosphorylation leads to fimbrin activation must be regarded as sub judice at present. Podosomes that participate in cell adhesion and locomotion have been found to contain other cytoskeletal linking proteins such as talin and vinculin, besides fimbrin. L-fimbrin has been shown to regulate integrin-mediated adhesion of leukocytes (S.L. Jones et al. 1998). It is possible that fimbrin is instrumental in the organisation of physical pathways, such as fimbriae and podosomes, by which extracellular signals for intercellular and cell-substratum adhesion are transduced to the cell.

Another membrane organelle with which fimbrin might be associated in organising a signal transduction machinery is the caveola. Caveolae are plasma membrane invaginations, of 50 to 100 nm dimension, that occur in many cell types. Caveolae have been attributed with many functions, notably transport of molecules across endothelia and signal transduction (Lisanti et al. 1995). A major component of caveolae is a 21- to 24-kDa protein called caveolin. Caveolin is said to function as a scaffolding protein that organises the signalling molecules in the caveolae. That caveolae contain essential components of the signal transduction machinery may be deemed to be firmly established. Thus caveolin has been shown to interact directly with signal transduction molecules such G-protein a subunits and the ras protein (Lisanti et al. 1995; Song et al. 1996). The receptors for PDGF and EGF are located in caveolin-rich microdomains, and caveolin has been shown to interact directly with the EGF receptor (G.X. Liu et al. 1996; Couet et al. 1997). The interaction of caveolin negatively regulates RTK activities associated with activated EGFr and c-erbB2 (Couet et al. 1997), and in mammary tumours expressing c-erbB2, caveolin expression is down-regulated (Engelman et al. 1998). Therefore, this interaction between growth factor receptors and caveolin may deregulate the transduction of the growth factor signals. Compatible with this view are recent reports that overexpression of caveolin results in growth inhibition of tumour cells (S.W. Lee et al. 1998; T. Suzuki et al. 1998). On the other hand, caveolin has been associated with cancer progression. It is reported to be overexpressed in infiltrating ductal carcinoma of the breast and prostate. In prostate cancer, caveolin is expressed in both primary and metastatic tumours (N. Yang et al. 1998). These two sets of data may appear to contradict each other, but it is possible to envisage caveolin functioning as a growth inhibitor in the early stages of tumour development. Caveolin might function in later stages of tumour progression merely by virtue of its ability to promote cell-cell and cell-substratum adhesion, which are essential requisites for a successful transition of the cancer cell along the metastatic cascade. In this connection, it would be worthwhile to note that T-cadherin, an adhesion-mediating molecule, also occurs in caveolin-rich plasma membrane microdomains (Philippova et al. 1998). After a seemingly long digression, it should also be noted that fimbrin is a component of the caveolae. Fimbrin occurs together with other proteins, e.g., src and ezrin, in cell membranes that are rich in caveolin (Mirre et al. 1996). The available evidence, albeit circumstantial in nature, may be interpreted as implicating fimbrin in tumor-igenesis and tumour progression by deregulating signal transduction pathways and cell adhesion mechanisms.

Regulation of Fimbrin Expression

The apparent tissue-specific distribution of fimbrin isoforms has led inevitably to investigations directed toward understanding the mechanisms of fimbrin expression and whether there is any tissue-specific regulation of its expression. C.S. Lin et al. (1997) have characterised the promoters of human and murine L-fimbrin. They described considerable similarity in the organisation and found that both promoters functioned with equal efficiency in most cell types. Therefore, the apparent tissue-specific and differentiation-related expression of fimbrin could be due to posttran-scriptional modification of fimbrin rather than to its regulation at the transcriptional level. Phosphorylation could be a mechanism of regulation of fimbrin function, as demonstrated for L-fimbrin. The serine residues of the head-piece region of L-fimbrin are phosphorylated (Messier et al. 1993), although earlier, Namba et al. (1992) found unphosphorylated L-fimbrin of human T cells to be quite effective in F-actin bundling. Shinomiya et al. (1995) reported that LPS stimulated the phosphorylation of L-fimbrin in macrophages. Phosphorylation occurred on amino acid residues at the N-terminal region close to the first Ca2+-binding domain. Shinomiya et al. (1995) also noticed that the phosphorylated region contained motifs that are phosphorylated by CK II, protein kinase A (PKA), and PKC, but not motifs specific for MAPK. Frederick et al. (1996) also have recently implicated PKC in fimbrin phosphorylation and have confirmed further that only serine, not tyrosine, residue is phosphorylated. The possible linkage of fimbrin function with phosphorylation is indicated by the ability of cytokines and phorbol esters, among other agents, to induce fimbrin phosphorylation. Polymorphonuclear leukocytes (PMN) stimulated by IL-8, IL-1, neutrophil-activating proteins, monocyte-derived neutrophil chemotactic factor, and TNF have been reported to stimulate the phosphorylation of I-fimbrin. Phosphory-lation was also influenced by phorbol 12-myristate 13-acetate (PMA) (Shiroo et al. 1988; Shibata et al. 1993a, 1993b). Fimbrin of T cells is phosphorylated in response to IL-2 (Zu et al. 1990). The adhesion of neutrophils to immune complexes induces L-fimbrin phosphorylation. Furthermore, it appears that phosphorylation-mediated regulation may be distinct from calcium-mediated regulation of fimbrin (Jones and Brown, 1996). It seems likely that the transduction of extracellular signals involves this important component of the membrane cytoskeleton. However, despite the clear demonstration of a fimbrin phosphorylation response to extracellular signals, there is little direct evidence that these features are functionally related. Messier et al. (1993) provide circumstantial evidence for this. They found phosphorylated fimbrin was mainly associated with the insoluble cytoskeleton. When this is read with their observation that the serine residues of the head piece were specifically phosphory-lated, one might be justified in concluding that phosphorylation could regulate the process of actin-binding and bundling by fimbrin.

Whether the isoforms have different functions in the regulation of cell morphology is a question that has been addressed, albeit superficially, by Arpin et al. (1994). In CV1 fibroblast-like cells, both T- and L-fimbrin cause change of cell shape and reorganisation of the actin stress fibres, and only L-fimbrin is associated with microfilaments. In epithelial cells such as LLC-PK1 cells, T-fimbrin remains associated with actin filaments of microvilli and produces shape changes in them. L-fimbrin has no effect on these structures. These observations might suggest functional differences between the isoforms. The invasion of Shigella flexneri, a bacterium that causes dysentery in humans, involves the formation of heavy actin polymerisation and actin bundling near the site of host cell contact with the bacterium. These bundles form protrusions with which the bacterium coalesces. Adam et al. (1995) experimentally overexpressed T- and L-fimbrin in HeLa cells and demonstrated that T-fimbrin might be preferentially recruited to the zone of bacterial entry.

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