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Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha- actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T- plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

cated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.
TIN filaments are commonly organized in the cytoplasm into bundles or gels. Actin bundles are closely spaced, parallel arrays of filaments that provide structural support for fingerlike extensions of the plasma membrane such as microvilli, stereocilia, and filopodia. This arrangement of filaments contrasts with the orthogonal lattice of filaments in an actin gel, which compose a subcortical scaffold associated with the plasma membrane. Actin bundles and gels are formed by separate classes of actin crosslinking proteins (Pollard and Cooper, 1986). Although these proteins must bind actin, it is unknown whether they share similar mechanisms of binding and cross-linking actin illaments.
Actin gelation proteins consist of a class of large, flexible multisubunit molecules that cross-link actin filaments into a loosely organized meshwork supporting the plasma membrane. This network is anchored to the membrane by integral and peripheral membrane proteins. Various actin gelation proteins, including alpha-actinin (Noegel et al., 1987;Blanchard et al., 1989), actin-binding protein (ABP) 1 (Hartwig and Stossel, 1975), filamin (Wang et al., 1975), spectrin (Morrow, 1989), and actin gelation protein (Condeelis et al., 1981;Noegel et al., 1989), have been identified based on their ability in vitro to increase the viscosity of actin ilia-1. Abbreviations used in this paper: ABE actin-binding protein; PMA, phorbol myristate acetate. ments. Actin gelation proteins are either dimers or tetramers. This oligomerization is required for actin cross-linking activity because monomeric actin-binding domains can bind but not cross-link actin filaments (Mimura and Asano, 1986). Gelation proteins have globular amino-and carboxyterminal domains that are separated by long stretches of repeated crossed-beta sheet or alpha-helical segments. Actin binding activity in alpha-actinin has been identified in the amino-terminal globular (250 residues) domain (Mimura and Asano, 1986). This domain has also been identified at the amino terminus of the gelation proteins and dystrophin (Hammonds, 1987;Davison and Critchley, 1988).
Because fimbrin and villin bundle actin in the same microvillus, we were interested in comparing fimbrin structure with villin. In this paper we describe the complete sequence of fimbrin deduced from full-length eDNA clones from chicken intestine. The sequence reveals that fimbrin is organized, like villin, into headpiece and core domains, but that the two proteins are unrelated in sequence. The fimbrin headpiece domain lies at the amino terminus and contains two calmodulin-like calcium-binding sites. The carboxyterminal core domain consists entirely of a fourfold repeat. Each pair of these repeats is homologous to the actin-binding domain of alpha-actinin and the putative actin-binding domains of dystrophin, actin gelation protein, and betaspectrin.
Fimbrin is also homologous to human L-and T-plastin, 68-kD polypeptides of unknown function. L-plastin is expressed in the cytoplasm of leukocytes (Goldstein et al., 1985;Lin et al., 1988Lin et al., , 1990 and in response to IL-1 and phorbol myristate acetate (PMA) acti-eation, L-plastin is phosphorylated at serine residues (Goldstein et al., 1985;Anderson et al., 1985;Matsushima et al., 1988). T-plastin is constitutively expressed in epithelial and mesenchymal cells derived from solid tissues but neoplastic cells from these tissues express both plastin isoforms. These results identify possible regulation of the membrane-associated cytoskeleton by phosphorylation of fimbrin and possible functional differences between fimbrin isoforms.

Fimbrin Proteolysis
Fimbrin was purified from the supernatant of calcium extracted chicken intestine epithelial cells using methods modified from Glenney et al. (1981b). The superuatant was passed through a DNase column to remove villin and actin. Fimbrin does not bind to the column and the flow-through fractions containing fimbrin were pooled. Fimbrin precipitated in a 45-60% ammonium sulfate cut and was further purified by gel filtration thorough AeA 34 and ion exchange chromatography on DEAE-Sephacel (Pharmacia Fine Chemicals, Piscataway, NJ) and AftiGel Blue Agarose (Bio-Rad Laboratories, Richmond, CA). The purified protein was desalted into 150 mM NaCI, 1 mM MgCI2, 0.1 mM EGTA, 10 mM Pipes buffer (pH 7.25) at a concentration of 1 mg/ml and digested with papain (1:400 wt/wt) at room temperature. Aliquots were removed at t = 5, 15, 30, 45, 60, 90, and 120 min and quenched by addition of PMSF to 0.1 mM and then boiled in SDS sample buffer. The digest products were separated by PAGE.

Amino-Terminal Sequence Analysis
Purified fimbrin was digested to completion with trypsin. The peptides were separated by reverse-phase HPLC, and well-separated peaks were collected manually and sequenced using a gas-phase instrument (model 470; Applied Biosystems, Inc., Foster City, CA) equipped for on-line analysis of phenylthiohydantoin (PTH) derivatives of amino acids. Fimbrin core generated by mild V-8 proteolysis was electroblotted onto an lmmobilon membrane (Millipore Corp., Bedford, MA) as described previously (Matsndaira, 1987) and the amino terminus was sequenced using a gas phase sequencer (model 2090E; Porton Instruments, Tarzana, CA) equipped for on-line PTH amino acid identification.

Construction of Chicken Intestine cDNA Library
RNA was isolated from chicken intestine epithelial cells by the guanidinium isothiocyanate method (Chirgwin et al., 1979), and poly(A) + RNA was isolated by oligo (dT)-cellulose column chromatography, eDNA was syn-thesized from 5 t~g of POly(A) + RNA with Moloney marine leukemia virus reverse transeriptase (Pharmacia Fine Chemicals) and random primers. Eco Rl/Not I linkers were attached and DNA was ligated into ),gtl0 and packaged in vitro (Gigapack II Plus; Strategene Corp., La Jolla, CA). The total library, unamplified, contained 1.9 × 106 recombinants.

Cloning and Sequencing of Fimbrin eDNA
A rabbit polyclonal antiserum (R163.3) raised against purified chicken intestine fimbrin was used to screen transformants (10 ~ clones) in a ~gtl I library constructed from chicken intestine poly(A)+ RNA (Bazari et al., 1988). A single clone (SW6) containing an internal ECO RI site was isolated, cloned into M13, and sequenced on both strands using dideoxy-chaln termination methods. This clone (2.4 kb) contained sequences that matched seven peptide sequences derived from V-8 and trypsin digests of chicken intestine fimbrin. SW6 did not contain the complete fimbrin sequence because its 5' coding region started in the middle of protein sequences corresponding to the NH2 terminus of fimbrin core. An 0.3 kb Xba I/Eco RI restriction fragment of clone SW6 was randomly labeled with 32p and used to screen 2x 105 recombinants plated from the XgtlO library. Nine positive clones were isolated. The clones were 1.0-3.7 kb in size and all contained an internal Eco RI site. Two clones (M38 and M48) were sequenced on both strands as described above and a 1.9-kb open reading frame corresponding to the fimbrin protein sequence was identified. The introns in clone M48 were sequenced on one strand. The fimbrin protein sequence was compared with the GenBank and NBRF data bases using TFASTA. Sequence homologies were further analyzed and aligned using BESTFIT (UWGCG, Devereux et al., 1984).

Northern Blot Analysis
Poly(A) + RNA (2 #g) isolated from chicken intestine epithelial cells was fractionated in formaldehyde-agarose gels and transferred to nylon membranes (Biodyne; Pall Corp,, Glen Cove, NY). The membranes were hybridized with randomly labeled 32p probes generated from restriction digests of M38, M48, or SWt, washed with 2x SSC and 0.1% SDS at room temperature, dried, and exposed to x-ray film. The size of the transcript was determined by comparison with 32P-labeled RNA size markers that were loaded in adjacent lanes.

Bacterial Expression of Piastin lsoforms
The corrected, full-length coding sequence of human L-or T-plastin (Lin et al., 1988(Lin et al., , 1990 was cloned into the recombinant plasmid pET-3C (Studier and Moffat, 1986) having the T7 RNA polymerase promoter and transfected into Escherichia coil, Expression was induced with isopropyl-~-D-thiogalactopyranoside, Lysates of uninduced and induced bacteria were solubilized with SDS sample buffer and subjected to one-dimensional gel electrophoresis and immunoblot analysis+

Autoradiography
Human SCC cells (gift from George Milo, Ohio State University) were labeled for 6 h with [3~S]methionine and subjected to two-dimensional gel electrophoresis as previously described (Lin et al., 1988). Proteins were electroblotted to nitrocellulose using a semi-dry blotting apparatus (Electrobiotransfer, Sunnyvale, CA) at a constant current (1.0 mA/cm 2 of gel) for 1 h. The filters were stained with Amido black to determine the effectiveness of protein transfer, incubated with a 1:I,000 dilution of rabbit anti-chicken fimbrin antiserum for 17 h, and subsequently incubated with peroxidase-conjugated goat anti-rabbit IgG (1:500 dilution; Bio-Rad Laboratories) for 2 h. The filters were developed to visualize the secondary antibody and exposed to x-ray film for 2 and 18 h to identify radioactive proteins.

Plastin lsoform Expression in Different Cell Lines
Various primary or secondary cell strains established from different tissues were assayed for L-and T-plastin expression by Northern blot analysis as described previously (Linet al., 1988) or by two-dimensional PAGE as described previously (Goldstein et al., 1985). Figure L Proteolysis of fimbrin produces two fragments, fimbrin core (58 kD) and headpiece (10 kD). With longer times of digestion, fimbrin core remains stable but headpiece is degraded into smaller fragments. Numbers on the left indicate the positions of the molecular weight standards.

Fimbrin Proteolysis
In nondenaturing conditions small amounts of papain ( Fig.  1), trypsin, thermolysin, proteinase K, subtilisin, or V-8 protease (not shown) cleaved intact fimbrin with the simultaneous appearance of two fragments, termed core and headpiece (58 and 10 kD). With longer times of digestion, the fimbrin headpiece but not core was cleaved to smaller fragments. Neither intact fimbrin nor fimbrin headpiece polypeptides could be sequenced, suggesting that the amino terminus of each was blocked and that fimbrin headpiece corresponded to the amino-terminal fragment of fimbrin. The amino terminus of fimbrin core was sequenced (see Fig. 4). The presence of a sequenceable amino terminus, size, and time course of appearance during proteolysis suggested the core fragment was derived from the carboxy-terminal part of fimbrin.

l~mbrin cDNA and Protein Sequence
A fimbrin-specific antiserum identified a single cDNA clone (SW6) in a hgtl 1 library (Fig. 2). This clone (2.4 kb) hybridized with a 3.2-kb band in Northern blots of chicken intestine RNA (Fig. 3). The sequence of SW6 contained an open reading frame encoding a 498-amino acid polypeptide. Two other clones (M38, 2.3 kb; M48, 3.7 kb, Fig. 2), isolated from the Xgtl0 library by screening with SW6 as a probe, contained a 1.9-kb open reading frame encoding a 630amino acid protein with a predicted molecular weight of 70,894 (Fig. 4). The coding regions of all three clones were identifical. One clone (M48) lacked a poly(A) tail and contained additional sequences in the 5' untranslated region (90 bp) and in the coding region (830 bp) that were not in M38 or SW6. The 830 bp sequence lies in the codon for cys165 between nucleotides 496 and 497. Because there are splicejunction sequences at the borders of the additional sequences, this clone probably represents an incompletely processed transcript. The amino terminus of the polypeptide was assigned based on the calculated molecular weight of the encoded protein and the absence of other initiation codons in the region. Fimbrin headpiece corresponded to residues 1-114 and fimbrin core to residues 115-630.

Duplicated Domains in Fimbrin and Plastin
A search of sequence data bases (GenBank and NBRF) showed that fimbrin is the chicken homologue of human T-and L-plastin. L-and T-plastin were cloned from a human fibroblast library (Lin et al., 1988), and encode 630 and 627 amino acid proteins respectively. The sequences of both isoforms have been recently amended (Lin et al., 1990) to include the correct amino termini. Fimbrin was slightly more homologous with T-plastin (72.1% identity in 627 residues)    (Table II) shows that the A regions are more similar to ~ regions than to B or B' regions. The percent identities between the regions are low (15-28%); however, each region contains a common pattern of conserved residues (boxed) within a 90-residue sequence (Fig. 6). This sequence is bounded by a conserved trp-x-asn sequence at the amino terminus and by a asp-ile/leu/val-val-x-x-pro sequence at the carboxy terminus. Most of the conserved residues are hydrophobic.

Homologies with Actin Gelation Proteins
The AB regions of fimbrin are homologous with the aminoterminal domains of chicken (Lemaire et al., 1988) and human (Koenig et al., 1988) dystrophin (18.7 and 19.8% identical) and chicken alpha-actinin ( Baron et al., 1987) 17.6% identical) ( Fig. 9 and Table I). Mimura and Asano (1986) demonstrated that the amino-terminal domain of alphaactinin binds actin filaments. Other recent reports have shown that the actin-binding domain of alpha-actinin is homologous with the amino-terminal domains of Drosophila beta-spectrin (Byers et al., 1989) and Dictyostelium actin gelatin protein (Noegel et al., 1989). These domains were 20.7 and 17.2% identical with the AB region of fimbrin. The amino-terminal domains of the actin gelation proteins also displayed the AB organization that we detected in fimbrin and plasfin. Most of the boxed areas in Fig. 6 indicate the residues conserved not only between fimbrin and plastin but also with the amino terminal domains of dystrophin and homologues. Other conserved areas, marked by the consensus sequence, are unique for the A and ~¢ domains or the B and B' domains. The consensus sequences shared by the A and B domains are primarily hydrophobic residues, clustered in two groups and separated by charged residues. The duplicated sequence within the amino-terminal domains of the actin gelation protein sequences have not been previously reported.

Calmodulin-like Calcium-binding Sites
The sequence of fimbrin headpiece shows no similarity to fimbrin core or to villin headpiece. A database search revealed that fimbrin headpiece is homologous with vertebrate calmodulin (Simmen et al., 1985) (34.7% identity) and a pseudogene (Stein et al., 1983) for skeletal muscle calmodulin (37.5 % identity). Although not expressed in muscle, the pseudogene sequence encodes a fully functional calmodulin, demonstrated by binding studies on protein expressed in bacteria . Matrix comparisons between headpiece and calmodulin (not shown) indicated that two regions in headpiece are homologous to calcium-binding sites of calmodulin. Previous sequence analysis of plastin had described a single calcium binding site (Lin et al., 1990). Both of the identified regions in headpiece contained a consensus calcium-binding site flanked by predicted alphahelix, forming a pair of EF hand or helix-loop-helix motifs (Fig. 7). In the three-dimensional structure of calmodulin, the calcium-binding domains I and II and domains HI and IV of calmodulin are at opposite ends of a central alpha-helical linker region (reviewed by Strynadka and James, 1989). Fimbrin headpiece is more similar to calmodulin domains III and IV (37.5% identity) than to domains I and II (23% identity). Both sites in fimbrin headpiece are predicted to

T T I S R E E L E E L R E A F N K I D I D N S G Y V S D Y E L Q D L F K E E R L S E E Q I A E F K E A F S L F R D G D G C I T T M E L G T V M R S L G Q N 10
30 40 flanking alpha-helices of calmodulin are indicated. Following the convention for describing calcium-binding sites, the positions of the residues that coordinate with calcium are labeled x, y, and z.

L P G K V R E I I E K I F A V T D S N K D G K I N F E E F V S L I Q E L K S P T E A E L Q D M V G E V D A D G S G T I D F P E F L S L M A R K M R
bind calcium, but their specificities and affinities can not be predicted from the sequence.

Fimbrin/Plastin Homology Demonstrated by Antibody Cross-Reactivity
Isoforms of plastin, identified by their positions on twodimensional gels, have been detected in a variety of human and mouse cells (Goldstein et al., 1985). Fimbrin has also been detected (using immunofluorescence) in a variety of cell types from different species (Bretscher and Weber, 1980). Fig. 8 a shows in an autoradiogram of [~sS]methioninelabeled proteins of the human squamous cell carcinoma cell line SCC separated by two-dimensional electrophoresis. L-plastin focused at a characteristic position (pI 5.9, 68 kD). When a blot of the two-dimensional gel (Fig. 8 b) was reacted with anti-fimbrin serum, L-plastin, and phospho-Lplastin were detected. T-plastin is less abundant in these cells and could not be detected on the two-dimensional gel or the  immunoblot. To show that our polyclonal antisera could detect T-plastin, we tested for cross-reactivity with recombinant T-and L-plastin (Fig. 8 c) on immunoblots. The fimbrin antisera crossreacted with recombinant L-and T-plastin polypeptides (Fig. 8 d) that were expressed in bacteria after induction with IPTG. Control lanes showed no crossreactivity with bacterial proteins.

Isoform-specific Expression
The presence of two fimbrin isoforms in mammalian cells may signify functional differences in actin-binding or cytoskeletal organization. To determine whether plastin isoform expression is restricted to particular cells, we surveyed a variety of human cell types (Table HI) for expression of L-and T-plastin isoforms using Northern blot analysis or two-dimensional gel electrophoresis. In normal cell strains assayed, expression of L-plastin was restricted to replicating populations of hematopoietic cells: erythrocytes and platelets exhibited no detectable L-plastin (data not shown). Cell strains derived from normal solid tissues expressed only T-plastin. Expression of the plastin isoforms in human cancer cells was studied using the same assays. Like their normal counterparts, T-and B-lymphoblastoid and myeloid cell lines expressed only L-plastin. However, in contrast with our findings for normal cells from solid tissues, 16 out of 22 cell lines derived from solid tumors or tumor-derived secondary cultures (not established as stable cell lines) expressed L-plastin in addition to T-plastin. In most cases, the L-plastin levels were several-fold higher than T-plastin although expression of L-plastin in individual tumor-derived cell lines varied greatly (data not shown) compared with the more constant level of T-plastin expression (Lin et al., 1988). The retinoblastoma cell line Y79, an anchorage-independent cell line, was the only human cell line tested that did not synthesize detectable levels of plastin messenger RNA or polypeptide (Lin et al., 1988).

Discussion
Our results provide the first evidence that links two classes of actin-binding proteins, actin bundling proteins and actin gelation factors, into a structurally related family of actin cross-linking proteins. A striking feature of these proteins is their modular organization (Fig. 9). A simple model of fimbrin depicts an amino-terminal calcium-binding domain and tandem arrangement of actin binding domains, AB and gB'. Each actin-binding domain consists of a duplicated sequence, suggesting that fimbrin structure arose from two gene duplication events. The first duplication resulted in the A and B domains; a second duplication produced the ?¢ and B' domains. ,The close spacing of actin filaments in a fimbrin bundle might result from the tandem arrangement of closely separated actin-binding domains.
Different arrangements of a single actin-binding domain ( Fig. 9) can account for the variety of actin gels and bundles present in a cell. The actin gelation proteins generally consist of an actin-binding domain, a rod-like spacer domain, and a carboxy-terminal domain responsible for binding calcium, membranes, or other actin-binding proteins. Alphaactinin for example, contains a single actin-binding domain per monomer, but when oligomerized into an antiparallel dimer the two actin-binding sites lie at opposite ends of the molecule. Proteins in which actin-binding domains are spaced increasingly apart from each other retain crosslinking activity but bundle actin filaments more loosely (Brotschi et al., 1978;Condeelis et al., 1984). The flexibility of a protein, increasing with length of the molecule, could allow the crosslinking of orthogonally arranged filaments into a three-dimensional meshwork.

Role of Calcium in Actin Bundle Formation
Fimbrin is composed of two structural domains (Fig. 9), headpiece and core, linked by a protease-sensitive region. Sequence homologies with other actin-or calcium-binding proteins suggest that these domains have separate functional roles: headpiece binds calcium and core binds actin. By equi-  Figure 9. A diagram of the modular arrangement of domains in fimbrin, plastin, alpha-actinin, dystrophin, beta spectrin, and actin gelation protein. Domains homologous to the AB or A'B' regions of fimbrin and plastin are indicated. Circles identify calmodulin-like calcium-binding domains. The boxed domains at the carboxy terminus of the gelation protein are responsible for membrane-binding or oligomerization of the polypeptide. librium dialysis, Glenncy and collaborators (1981) identified in fimbrin a single calcium-binding site that is competed by Mg :+. This site is probably one of the sites predicted in timbrin headpiece; whether a second calcium-binding site in headpiece also binds calcium is not known. The functional role of calcium in fimbrin regulation is not understood because preliminary evidence (Glenney et al., 1981b;Matsudaira, E, manuscript in preparation) suggests that fimbrin binding to actin is independent of calcium. The homology between fimbrin and plastin is weakest in the headpiece domain raising the possibility that the isoforms may be differentially regulated by calcium. Alpha-actinin, which has actinand calcium-binding domains (Noegel et al., 1987) similar to fimbrin, is inhibited by calcium from crosslinking actin filaments. Chemical cross-linking studies demonstrate that the calcium-binding domain interacts with the actin-binding domain (Imamura et al., 1988). Although fimbrin headpiece is more closely related to calmodulin (34.7% identify) than the calcium-binding domains of alpha-actinin (18.4% identity), a similar interaction of headpiece with core may influence actin-binding activity in a different fimbrin isoform.
The calcium-binding headpiece domains of fimbrin and villin are not homologous despite their similar sizes. In villin this domain binds calcium (Hesterherg et al., 1985) and lies at the carboxy terminus (Glenney et al., 1981b). The isolated villin headpiece domain binds but does not bundle actin filaments and binding is not affected by calcium. We do not know if fimbrin headpiece contains an actin-binding activity.

Do Conserved Sequences Represent Actin-binding Sites?
The presence of a consensus sequence in various actin binding proteins is often interpreted to indicate conservation of an actin-binding site, given the evolutionary conservation of actin (Blanchard et al., 1989). An alternative interpretation of a conserved sequence in actin-binding proteins is suggested by the fact that the most variable region of the actin sequence is at the acidic amino terminus, where basic residues in several actin-binding proteins can be chemically crosslinked (Sutoh and Mabuchi, 1986). The consensus sequence in the A and B domains of fimbrin and the larger actin binding proteins is largely hydrophobic and thus is likely to be buried in the protein, an unlikely location for an actinbinding site.
A possible function for the conserved hydrophobic sequence is suggested from studies on villin and other actinsevering proteins with a conserved hydrophobic repeated se-quence. We have concluded that these sequences might be involved in maintaining a conformation or structure of the domains (Matsudaira and Janmey, 1988): the consensus sequence maps within protease-resistant regions of villin, and amino acids such as cysteine (Matsudaira et al., 1985) or methionine (unpublished data) within the conserved repeat cannot be chemically modified in the folded, native protein. These experiments directly support the prediction that conserved sequences in villin are buried in the protein. Although we have not mapped the residues that are accessible to solvent and the protease-resistant domains of fimbrin to the same resolution as viUin, we suggest the conserved sequences in fimbrin may possibly play a similar structural role.

Possible Regulation of Actin Binding by Phosphorylation
The identification of fimbrin as plastin suggests that the actin-and/or calcium-binding activity of fimbrin may be modulated by phosphorylation. Two other actin bundling proteins, band 4.9 of the erythrocyte membrane (Husain-Chistri et al., 1988) and synapsin I of the nerve synapse (Bahler et al., 1986), also become phosphorylated. In both cases, phosphorylation inhibits actin bundle formation. L-plastin was first identified on the basis of its exclusive expression in transformed cells and was later detected in normal lymphocytes and macrophages. L-plastin becomes phosphorylated, presumably by protein kinase C, on unidentified serine residues when leukemia cells are activated by PMA or when leukocytes are activated by IL 1 (Goldstein et al., 1985;Anderson et al., 1985;Matsushima et al., 1988). The level of phosphorylation varies between cell types, but can approach as much as 30% of the total soluble L-plastin (Leavitt, J., unpublished data). Unlike L-plastin, T-plastin phosphorylation has not been detected in any cell line and thus T-plastin might not he a substrate for kinases. Fimbrin purified from chicken intestine epithelia is a single spot on a two-dimensional gel and phosphorylated intestinal fimbrin has not been detected.

Functional Differences between Fimbrin lsoforms
In addition to differences in sequence and phosphorylation, the differential expression of the L-and T-isoforms in hematopoietic and non-hematopoietic cells and the change in isoform expression in neoplastic cells suggest the isoforms are functionally different. This differential expression was not detected in immunofluorescence studies of various cells (Bretscher and Weber, 1980a) because polyclonal antisera raised against chicken fimbrin cannot distinguish the two human fimbrin/plastin isoforms (Fig. 8 d). The pattern of isoform expression may be correlated with the motile or adhesive properties of the cell because antibodies specific for fimbrin have detected fimbrin in focal contacts and adhesion sites on the ventral surface of cultured ceils, monocytes, and macrophages (Bretscher and Weber, 1980b;Carley et al., 1985). Recent identification of protein kinase C at sites of focal contact Oaken et al., 1989) and our finding that fimbrin is a cytoplasmic phosphoprotein in leucocytes raise the tantalizing possibility that fimbrin phosphorylation is involved in cell-substratum or cell-cell interactions. Changes in expression and distribution of microfilament proteins often accompany neoplastic transformation in many cell systems. The discovery of phosphorylated isoforms of fimbrin provides additional molecular details of the possible mechanism of regulation of cellular actin bundles during the re-organization of the microfilament-based cytoskeleton.