|
|
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© The Rockefeller University Press,
0021-9525/1999//831 $5.00
The Journal of Cell Biology, Volume 146, Number 4,
, 1999 831-842
Original Article |
Integrating the Actin and Vimentin Cytoskeletons
: Adhesion-Dependent Formation of Fimbrin–Vimentin Complexes in Macrophages
b Department of Biology and Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142
c Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60208
Whitehead Institute of Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142.(617) 258-6691(617) 258-5206
correia{at}wi.mit.edu
Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH2-terminal domain of vimentin and the vimentin binding site at residues 143–188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin–vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.
Key Words: fimbrin • vimentin • cytoskeleton • adhesion • microfilaments
© 1999 The Rockefeller University Press
THE intricate structure of the microfilament (MF),1 microtubule (MT), and intermediate filament (IF) cytoskeletons is organized by an assortment of accessory proteins that stabilize or cross-link filaments or attach filaments to the membranes of organelles and the cell surface (Schliwa 1986; Bershadsky and Vasiliev 1988). In most cases, we know much about the function of the binding proteins within one cytoskeletal system; for example, the role of actin cross-linking proteins in bundles and networks of actin filaments (Matsudaira 1991, Matsudaira 1994). In contrast, relatively little is known whether or how the coassembly and coorganization of cytoskeletal systems are integrated and coordinated. The very earliest work used MT and IF depolymerizing drugs to demonstrate that the organization of the two cytoskeletal systems are interdependent; the disassembly of one disrupts the pattern of the other (Goldman and Knipe 1972; Croop and Holtzer 1975; Blose and Chacko 1976; Wang and Choppin 1981). More recent studies have identified several proteins including the IF-associated protein, plectin, that could serve as physical cross-links between the different cytoskeletal systems (Svitkina et al. 1996). In addition to biochemical demonstration of binding, electron micrographs of cultured fibroblast cells show plectin molecules bridging MTs and vimentin filaments. However, the story is likely to be more complicated because plectin is also implicated in interactions with the actin cytoskeleton based on the presence of an actin-binding domain (ABD) in the protein sequence (Wiche et al. 1991, Wiche et al. 1993; Liu et al. 1996). Thus, it is possible that cytoskeletal-binding proteins may interact with several cytoskeletal systems.
An emerging model is that a superfamily of actin-binding proteins containing a calponin homology (CH) domain may play an important integrative function in controlling the organization of the major cytoskeletal systems in the cell. In most cases, this CH domain is duplicated to form the classic ABD of fimbrin, 
-actinin, spectrin, filamin, dystrophin, other actin cross-linking proteins, and the IF-binding protein, plectin (Dubreuil 1991; Hartwig and Kwiatkowski 1991; Otto 1994). In contrast, a single CH domain is found at the NH2 terminus of small G-protein regulatory molecules, vav and IQ GAP (Castresana and Saraste 1995; Brill et al. 1996). The list of functions attributed to these proteins implicate the CH domain in cross-linking MFs into bundles and networks and tethering IF and signaling proteins to the actin cytoskeleton (Castresana and Saraste 1995). Thus, CH domain proteins may directly control the structural organization of the cytoskeleton through one or more signaling pathways.
The experiments reported in this paper describe a new type of interaction between a subunit of the vimentin IF and an actin cross-linking protein, fimbrin. Fimbrin is a 68-kD actin cross-linking protein associated with actin bundles (Matsudaira and Burgess 1979; Bretscher and Weber 1980) in microvilli on the cell surface and in membrane ruffles, microspikes, and cell adhesion sites at the cell–matrix interface (Bretscher and Weber 1980; Marchisio et al. 1987; Matsudaira 1994). Conserved in sequence from yeast to humans, fimbrin consists of an NH2-terminal 12-kD calcium-binding domain followed by a pair of 27-kD actin-binding domains (ABD1 and ABD2) (Matsudaira 1991). Based on sequence and structure from x-ray crystallography, the ABDs consist of a pair of CH domains (de Arruda et al. 1990; Goldsmith et al. 1997). In mammals, three fimbrin isoforms are expressed in a tissue-specific pattern. The intestine-specific isoform, I-fimbrin, is localized to the brush border of intestinal and kidney epithelia (Lin et al. 1994). In contrast, the leukocyte-specific isoform, L-fimbrin, and the general isoform, T-fimbrin, are localized to cell–substratum adhesion sites in macrophages and fibroblasts and to the basal surface of differentiating intestinal enterocytes (Lin et al. 1993; Chafel et al. 1995). The adhesion site localization of fimbrin is curious because it does not label the actin bundle of the stress fiber but instead the site where the bundle is associated with cell–matrix adhesion proteins. This location at the base of the cell suggests a different function for the protein, possibly as a component of a cell adhesion complex involved in establishing cell polarity during embryogenesis. An important clue to the mechanism on how fimbrin is targeted to the base of the cell may be revealed by the identity of proteins that coprecipitated with fimbrin in adherent macrophages (Messier et al. 1993). The results in this paper show that fimbrin is complexed with vimentin subunits rather than vimentin filaments and both proteins colocalize at filopodia, retraction fibers, and podosomes on the ventral surface of cultured macrophages. The association between the two proteins is adhesion-dependent, implicating a mechanism that coregulates assembly of the two cytoskeletal systems at sites of cell adhesion.
 |
Materials and Methods |
|---|
 TopAbstract Materials and Methods ResultsDiscussionReferences |
|---|
Recombinant Proteins
Recombinant vimentin was expressed and purified by published protocols (Chou et al. 1997). Mutagenesis was carried out on cloned human vimentin in the pTZ18U vector (Muta-Gene Phagemid In Vitro Mutagenesis kit; Bio-Rad Laboratories). The 102C vimentin clone was generated by the deletion of the sequence encoding the NH2 terminus of vimentin and by the introduction of a new initiation ATG codon immediately preceding the codon for asparagine-102. For the N410 vimentin clone, a stop codon was introduced after the codon for isoleucine 410. Both constructs were subcloned into the PET-7 vector for expression in Escherichia coli.
For fimbrin expression and purification, BL21(DE3) cells were transformed and the cells were plated onto ampicillin plates. Colonies were inoculated into 2 liters of 2x YT media and shaken for 24 h at 37°C. The cells were harvested by centrifugation (4,000 rpm for 30 min) and suspended in 50 mM Tris, pH 8.0, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, and 25% sucrose, and frozen. The cell suspension was thawed, lysed by sonication, and centrifuged at 18,000 rpm for 30 min. The supernatant was dialyzed into 10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 1 mM sodium azide and loaded onto a DEAE-Sepharose fast flow column (Pharmacia Biotech) preequilibrated with the same buffer. After washing to remove unbound material, the column was eluted with a linear gradient of NaCl (0–300 mM). Peak fractions were pooled, concentrated, and loaded onto a Q-Sepharose column (Pharmacia Biotech) that was equilibrated in 10 mM Pipes, pH 7.0, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 0.1 mM PMSF, and 0.1 mM sodium azide. The protein was again eluted with a linear salt gradient (0–300 mM NaCl), pooled, and loaded onto a Sephacryl S200 HR gel filtration column (Pharmacia Biotech). The purified protein was concentrated on a microconcentrator (30-kD cut-off).
Glutathione-S-transferase (GST) fusion constructs were generated by PCR cloning of human L-fimbrin. cDNA regions corresponding to L-fimbrin protein sequences 97-248, 243-385, 97-188, 97-142, 143-188, and 119-160 were amplified using synthetic oligonucleotide primers (Life Technologies, Inc.). A nucleotide tail coding for an in-frame BamHI restriction site was engineered into each 5' PCR oligo; a nucleotide tail coding for an EcoRI restriction site and a downstream amber stop codon (TAG) was engineered into each 3' PCR oligo. PCR-amplified DNAs were subcloned, via the engineered BamHI and EcoRI restriction sites, into a GST-vector, pGEX-4T2 (Pharmacia Biotech), directly downstream of the GST transcription region. GST-fimbrin fusion proteins were produced by inducing log phase DH5- bacteria containing the GST construct with isopropyl-β-D-thiogalactopyranoside for 1.5 h, purifying the recombinant protein with glutathione agarose beads, and concentrating the eluate with a centriprep/centricon concentrator (Amicon).
Cell Culture
The cells used in these experiments were mouse macrophage cell lines P388D1 (ATCC TIB-63) and IC-21 (ATCC TIB-186). The cell lines were cultured in 85% RPMI-1640 medium, 15% FBS, and antibiotics (50 IU penicillin and 50 µg/ml streptomycin). Cell culture and maintenance techniques were performed as described by American Type Culture Collection.
Cell Extraction and Immunoprecipitation
P388D1 cells were grown to confluence (adherent cells), washed once with PBS, scraped off the plate in PBS, and pelleted by centrifugation. P388D1 cells were also grown in suspension (nonadherent cells), washed twice in PBS, and pelleted by centrifugation. The cells were lysed on ice in 10 mM Pipes buffer, pH 6.8, containing 0.5% Triton X-100, 300 mM sucrose, 100 mM KCl, 3 mM MgCl2, 10 mM EGTA, 2 mM PMSF, and 50 µM sodium vanadate (Messier et al. 1993). After 3 min, the cells were centrifuged at 10,000 rpm for 10 min. Aliquots of the supernatant representing the Triton-extractable fraction were precleared with protein A conjugated to agarose beads. For immunoprecipitation of fimbrin, the precleared supernatants were incubated with different antifimbrin sera (736.5, 738.5, 739.5, and 163.3) or their respective prebleeds for 1 h. For immunoprecipitation of vimentin, the precleared supernatants were incubated with a combination of antivimentin sera (Chemicon International, Inc.; Sigma Chemical Co.). Protein A agarose was added and the extracts incubated for 1 h. The beads were washed four times with buffer A (0.1% Triton-X 100, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0) and directly solubilized in the appropriate buffer for one- or two-dimensional analysis.
Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis was carried out as described by O'Farrell et al. 1977. Isoelectric focusing was carried out in the first dimension by using ampholytes pH 3-10 (Bio-Rad Laboratories), and setting a pH gradient from 4–8. Proteins were resolved in the second dimension on a 7.5% SDS–polyacrylamide gel. The antifimbrin complex immobilized on protein A agarose beads was solubilized in SDS-IEF buffer (0.5% SDS, 9.5 M urea, 2% ampholytes pH 3-10, and 5% 2-mercaptoethanol). After 10 min, an equal aliquot of Garrel's buffer (4% NP-40, 9.5 M urea, 2% ampholytes pH 3-10, and 5% 2-mercaptoethanol) was added and incubated at 37°C for 15 min. Samples were loaded at the basic end and allowed to electrophorese for 8,000 Vh. After isoelectric focusing was complete, the IEF gels were equilibrated in SDS sample buffer, and then electrophoresed in the second dimension. The gels were finally silver stained (Shevchenko et al. 1996).
Protein Identification
The silver-stained spots were excised from the gel, washed, and processed as described by Shevchenko et al. 1996. The protein was reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The gel piece was finally dried in a Speed-Vac and rehydrated at 4°C in digestion buffer (50 mM NH4HCO3) containing trypsin (Boehringer Mannheim). Excess trypsin was removed and the gel incubated overnight at 37°C. Digested peptides were extracted with 5% formic acid and 50% acetonitrile. The pooled extracts were dried, dissolved in 5 µl of 5% formic acid and 50% acetonitrile, mixed in a 1:1 ratio with either -cyano-4-hydroxy-trans-cinnamic acid or 3,5 dimethoxy-4-hydroxy cinnamic acid (Aldrich Chemical Co.), and then spotted onto the target plate. All mass spectra were obtained on the Voyager Elite mass spectrometer (DE-STR; PerSeptive Biosystems) operated in the reflector mode. A list of monoisotopic peptide masses (>1,000 and <3000 Da) was imported into PepFrag and MS-Fit that searched the Swiss-Prot or GenPept databases. The following parameters were used in searches: cysteines were modified by aminocarboxymethylation, proteolytic digestion with trypsin (3–4 incompletes), mass tolerance of 1 Da, and molecular weight range of 10 kD.
In other experiments, proteolytic fragments of fimbrin and vimentin were identified by microsequencing of electroblotted fragments following previously published protocols (Matsudaira 1987). Peptides were sequenced using a microsequencer (model 477A; Applied Biosystems).
Overlay Binding Assays
The fimbrin and vimentin concentrations were obtained by measuring absorbance at 280 nm and by SDS-PAGE. Protein was mixed with Sulfo-NHS-Biotin (Pierce Chemical Co. ) at a molar ratio of 1:20 and coupled following the supplier's protocol. Unreacted biotin was removed and the derivatized protein concentrated on a 30-kD cut-off microconcentrator (Amicon). Typically, 100% of the molecules were derivatized as assayed by binding to streptavidin-agarose.
The overlay binding assay was performed essentially as described by Merdes et al. 1991. Electrophoresis of protein samples was performed in duplicate; one of the gels was stained and the other electroblotted onto polyvinylidene difluoride (PVDF) (Millipore Corp.). The blot was blocked with 1% nonfat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% Tween 20) for 1 h. For the fimbrin overlay assay, the blot was incubated overnight with labeled fimbrin in 1% nonfat dry milk in TBST. For the vimentin overlay assay, the blot was incubated overnight with labeled vimentin in 1% nonfat dry milk in a low ionic strength buffer (8.03 mM NaH2PO4, 1.47 mM KH2PO4, 0.2 mM CaCl2 and 1% Triton X-100, pH 7.2). The blot was washed with TBST, incubated for 1 h with the ABC elite vector reagent (Vector Labs, Inc.), washed again with TBST, and finally developed using the ECL kit (Amersham).
In Vitro Binding with Soluble Vimentin
Purified vimentin was purchased (Cytoskeleton), whereas recombinant fimbrin and vimentin were expressed and purified as described above. Vimentin was kept in an unpolymerized state by disassembling filaments in 8 M urea, 5 mM sodium phosphate, pH 7.2, 0.2% 2-mercaptoethanol, and 1 mM PMSF. The sample was dialyzed overnight against 5 mM sodium phosphate, pH 7.4, containing 0.2% 2-mercaptoethanol and 0.2 mM PMSF. The protein concentration was adjusted to 
1 mg/ml and incubated with precleared fimbrin (50 µg each) at 37°C for 1 h in 1 ml of low ionic strength buffer containing 8.03 mM NaH2PO4, 1.47 mM KH2PO4, 0.2 mM CaCl2 and 1% Triton X-100, pH 7.2. Antifimbrin or preimmune serum was added and the proteins were incubated for an additional 1 h. Protein A–Sepharose was added for 1 h and the beads were finally washed three times with buffer A and directly solubilized in SDS–sample buffer. The samples were resolved on SDS-PAGE, electroblotted onto a PVDF membrane, and probed with an antivimentin HRP-linked antibody (Affinity Biologicals Inc.).
Polymerization of Vimentin and Binding Assay with Fimbrin
Vimentin was diluted to a concentration of 0.5 mg/ml in a total volume of 100 µl using assembly buffer (6 mM phosphate buffer, pH 7.4, 3 mM KCl, 0.2% 2-mercaptoethanol, 0.2% PMSF). Polymerization was achieved by adding 3 µl of 5 M NaCl and incubating at 37°C for 1 h. For cosedimentation assays, preformed vimentin IFs were incubated for an additional hour with fimbrin maintained at concentrations from 0.25 to 1 mg/ml. In other experiments, vimentin and fimbrin were mixed together and IF polymerization, then initiated in the assembly buffer. The proteins were collected by ultracentrifugation at 100,000 g for 30 min and analyzed by SDS-PAGE.
Estimation of Kd
Purified vimentin (2.5 µg) was applied to nitrocellulose membranes. The blots were placed into wells of a 12-well tissue culture plate (Costar Corp.) and an overlay assay with labeled fimbrin (0.1–1 µM) was performed as described earlier. The amount of labeled protein that bound was estimated by densitometric scanning (GS-700 Densitometer; Bio-Rad Laboratories) and quantified using the molecular analyst software (Bio-Rad Laboratories). Nonspecific binding of biotinylated fimbrin was determined by measuring the amount of binding to nitrocellulose containing no vimentin. Specific binding was determined by subtracting nonspecific binding from the total binding. Scatchard analysis was carried out to estimate the Kd of binding. Plot shown gives on the vertical axis bound/free fimbrin and on the horizontal axis bound fimbrin (pmols). The amount of fimbrin that bound in pmols was obtained from a standard curve of known amounts of biotinylated fimbrin directly spotted onto nitrocellulose.
Fimbrin Proteolysis
Small amounts of Lys-C (0.25 µg) were incubated with purified fimbrin (50 µg) under nondenaturing conditions in 50 µl of 25 mM Tris, pH 7.7, and 1 mM EDTA. Aliquots were removed at 1, 10, 15, and 30 min, quenched by the addition of PMSF, and then boiled in SDS–sample buffer. The digested products were resolved on SDS-PAGE gels that were either silver stained or electroblotted onto PVDF.
Immunofluorescence Microscopy
For immunofluorescence studies, cells were allowed to attach onto glass coverslips (VWR Scientific) for 1–48 h, gently rinsed in PBS, and fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After three washes with PBS, the cells were permeabilized by incubating with 0.1% Triton X-100 in PBS for 2 min before immunostaining. To prepare detergent-extracted cells, adherent cells were first incubated with ice-cold 10 mM Pipes buffer, pH 6.8, containing 0.5% Triton X-100, 300 mM sucrose, 100 mM KCl, 3 mM MgCl2, 10 mM EGTA, 2 mM PMSF, and 50 µM sodium vanadate for 3 min, washed with the same buffer, and then fixed with 4% PFA.
Nonspecific protein absorption was inhibited by incubating the cells for 1 h in PBS containing 3% BSA, 0.2% Tween 20, and 0.2% fish gelatin. Coverslips were incubated for 1 h at 37°C with affinity-purified rabbit–fimbrin antibody (737.4a) diluted 1:100 and goat–vimentin antibody (Sigma Chemical. Co.; Chemicon International, Inc.) diluted 1:50. The coverslips were washed with PBS and the cells were incubated for 1 h at 37°C with secondary antibodies, FITC-conjugated donkey anti–rabbit and Texas red–conjugated donkey anti–goat (Jackson ImmunoResearch Laboratories). After three washes with PBS, the coverslips were directly mounted in Vectashield (Vector Labs, Inc.) and examined. Cells examined for actin distribution were stained with rhodamine phalloidin (Molecular Probes Inc.).
Cells were imaged by epifluorescence microscopy using a Nikon TE300 microscope with 60 and 100x oil immersion lenses. The images were recorded with a Hammatsu Orca CCD camera and analyzed with the Open Lab software program (Improvision Inc.). For deconvolution microscopy, the cells were viewed on a Nikon Eclipse 800 fluorescence microscope with a 100x oil immersion lens. Image stacks were recorded at 134-nm intervals (z series) with a Hammatsu Orca CCD camera and analyzed with the CellScan software (Scanalytics) or Metamorph imaging system (Universal Imaging Corp.) using the exhaustive photon reassignment algorithm. The images were reconstructed in three dimension using either the acquisition or Imaris software (BitPlane). Colocalized proteins were identified by using the colocalization algorithm in Imaris, where voxels containing the two different signals were extracted. The images were finally processed with Adobe Photoshop.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Characterization of Fimbrin–Vimentin Binding
Because the fimbrin–vimentin complex is found in a Triton X-100 extract of adherent cells, we also investigated whether the complex was present in nonadherent cells. Although the total amount of vimentin in adherent and nonadherent cells is identical, we could immunoprecipitate a detergent soluble pool of vimentin only from adherent cells using vimentin-specific antibodies (Fig. 2). Furthermore, a fimbrin-specific antibody detected fimbrin only in vimentin immunoprecipitates from adherent cells. Reciprocal immunoprecipitations with fimbrin-specific antibodies confirmed the presence of an extractable complex in adherent cells but not in nonadherent cells (not shown). This extractable fraction of vimentin in adherent cells was estimated to be 1% of the total protein.
|
|
|
|
|
|
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The association between fimbrin and vimentin, reported in this study, is a new finding. Based on previous studies, fimbrin was shown to bind and cross-link actin filaments. This function was consistent with its localization in actin bundles that support cell surface microvilli (Bretscher and Weber 1980; Matsudaira 1991). However, early studies also detected fimbrin at the cell–matrix interface (Bretscher and Weber 1980; Marchisio et al. 1987), which suggested that fimbrin had a function associated with cell adhesion. This suggestion was reinforced by our finding that during embryogenesis L-fimbrin is transiently expressed and localized to the basal surface of embryonic enterocytes, whereas T- and I-fimbrin and the majority of F-actin are in the developing brush border at the apical surface of the same cells (Chafel et al. 1995). Because IFs are now recognized components of focal adhesions (Green et al. 1986; Bershadsky et al. 1987; Geiger et al. 1987; Seifert et al. 1992), interactions with fimbrin could explain how vimentin is differentially targeted to cell adhesion structures where it can assemble into IFs. In addition, association with vimentin that may be transported to adhesion sites in a microtubule-dependent mechanism (Prahlad et al. 1998; Yoon et al. 1998) can explain how fimbrin is targeted to adhesion sites where it can cross-link actin filaments into a bundle.
The fimbrin–vimentin interaction is unusual because fimbrin binds a vimentin subunit and not a filament. Typically, cytoskeletal networks are cross-linked by proteins that bind polymerized filaments (Svitkina et al. 1996; Yang et al. 1996). For example, elegant immuno EM studies show plectin cross-bridges between assembled IFs and MTs (Svitkina et al. 1996). Plectin is also implicated in binding to actin filaments through an actin-binding domain located at the NH2 terminus, whereas the IF-binding domain is found at the COOH terminus (Wiche et al. 1993; McLean et al. 1996). However, this is not the case for fimbrin. Its interaction with a subunit of vimentin is suggested by the following: the 1:4 stoichiometry of binding (Fig. 3 B), the absence of substantial fimbrin colocalization with vimentin filaments in the cell body, and the inability of fimbrin to bind polymerized vimentin in pelleting assays (Correia, I., and P. Matsudaira, unpublished data). The detergent-extracted fraction most likely includes the pool of unassembled vimentin subunits in the cell (Soellner et al. 1985) that are believed to be tetramers of vimentin. The identity of the vimentin subunit as a binding partner of fimbrin is also consistent with the low percentage (1%) of this species in the supernatant (Fig. 2). Thus, the interaction with a vimentin subunit and not a filament suggests that the complex has a function other than to cross-link the actin and IF cytoskeletons.
Binding to vimentin subunits places fimbrin in a unique position to influence the assembly and organization of both the actin and IF cytoskeletons. We speculate that the fimbrin–vimentin complex plays an early role in the assembly of the actin and vimentin cytoskeleton in filopodia and podosomes. This speculation is supported by our biochemical observation that the fimbrin–vimentin interaction in P388D1 cells is adhesion-dependent. The time course of the localization of fimbrin and vimentin in these structures, obtained by epifluorescence microscopy, also suggests that the complex is a transient structure that is involved early in cell adhesion. During the early stages of attachment of motile IC-21 cells, we detected costaining of fimbrin and vimentin in podosomes of expanding lamella. After 3 h, fimbrin does not become associated with the vimentin network of filaments (Fig. 7 and Fig. 8). Because fimbrin does not bind assembled vimentin filaments in vitro other proteins may assist in anchoring the vimentin network at mature adhesion sites. These observations are relevant as studies obtained from time-lapse and interference reflection microscopy indicate a preformed complex of cytoskeletal proteins that nucleate the recruitment of adhesive molecules before anchoring the lamellipodia and filopodia to the substratum (De Pasquale and Izzard 1987; Izzard 1988). Although the compositions of the nucleating proteins were not described, the fimbrin–vimentin complex is in the right place and at the right time to play this role.
This model of fimbrin–vimentin function is compatible with earlier reports from Tao and colleagues (Mabuchi et al. 1997), who described a similar interaction between desmin tetramers and calponin in smooth muscle cells. They proposed the idea that an IF subunit and an actin-binding protein may be involved in the assembly of IFs at dense bodies. We agree with their interpretation. In fact, it is alluring to consider that the interactions involve cell adhesions. As discussed in the following section, an interaction between an IF protein, desmin, or vimentin, and a protein with a CH domain, calponin, or fimbrin may represent a highly conserved function that may be related to the establishment of cell adhesion structures.
The fimbrin–vimentin complex is biochemically distinguished from the rest of the actin and IF cytoskeletons by its extraction properties. The critical observation is that fimbrin and vimentin are extracted from a subset of structures (podosomes, filopodia, and retraction fibers) by detergent. Biochemical quantitation shows the detergent-extracted population represents 1% of the total vimentin in the cell. Most vimentin is in a detergent-insoluble fraction, and fluorescence microscopy localizes the insoluble vimentin to filaments in the cell body and around the nucleus. Thus, the vimentin–fimbrin complex found in early cell adhesion structures is biochemically and structurally distinct from the vimentin network in the cell body. This distinction may provide useful approaches for studying the function of the complex in live cells.
The biochemical and structural properties of the fimbrin–vimentin complex may be explained by the location of fimbrin and vimentin binding sites on the proteins. Our overlay binding assays now tentatively map the region of fimbrin–vimentin interaction to residues 143–188 of the NH2-terminal actin-binding domain of fimbrin and the head domain of vimentin. These sites lie within interesting regions of both proteins. Fimbrin binds a pair of actin filaments using actin-binding domains that are located in the NH2- and the COOH-terminal regions of the molecule (Hanein et al. 1997). Quantitative modeling of a fimbrin x-ray structure to an EM reconstruction of fimbrin bound to actin filaments predicts that actin binds fimbrin through interactions at the two CH domains in the NH2-terminal actin-binding domain, ABD1 (Hanein et al. 1998). Vimentin also binds fimbrin on ABD1, opposite to the actin-binding site, but on the same side where the regulatory calcium-binding domain (residues 1–100) and the COOH-terminal actin-binding domain (ABD2) are located (Fig. 10). This location poses interesting implications for actin bundling. Although we have not yet studied the ability of the fimbrin–vimentin complex to bind and cross-link actin filaments, we suspect that vimentin binding to fimbrin may interfere with the ability of fimbrin to cross-link actin.
|
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grants to P. Matsudaira and R. Goldman and was conducted at the W.M. Keck Foundation Microscopy Facility at the Whitehead Institute.
1.used in this paper: ABD, actin-binding domain; CH, calponin homology; F-actin, filamentous actin; GST, glutathione-S-transferase; IF, intermediate filament; MF, microfilament; MT, microtubule; PVDF, polyvinylidene difluoride
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bershadsky A.D., Tint I.S. & Svitkina T.M.. Association of intermediate filaments with vinculin-containing adhesion plaques of fibroblasts, Cell Motil. Cytoskelet, 8, 1987, 274–283.[Medline]
Bershadsky A.D. & Vasiliev J.M., Cytoskeleton, 1988, Plenum Publishing Corp, New Yorkpp. 298 pp.
Blose S.H. & Chacko S.. Rings of intermediate (100 A) filament bundles in the perinuclear region of vascular endothelial cells. Their mobilization by colcemid and mitosis, J. Cell Biol, 70, 1976, 459–466.
Bretscher A. & Weber K.. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures, J. Cell Biol, 86, 1980, 335–340.
Brill S., Li S., Lyman C.W., Church D.M., Wasmuth J.J., Weissbach L., Bernards A. & Snijders A.J.. The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases, Mol. Cell Biol, 16, 1996, 4869–4878.[Abstract]
Castresana J. & Saraste M.. Does Vav bind to F-actin through a CH domain?, FEBS (Fed. Eur. Biochem. Soc.) Lett, 374, 1995, 149–151.
Chafel M.M., Shen W. & Matsudaira P.. Sequential expression and differential localization of I-, L-, and T-fimbrin during differentiation of the mouse intestine and yolk sac, Dev. Dyn, 203, 1995, 141–151.[Medline]
Chou Y.H., Skalli O. & Goldman R.D.. Intermediate filaments and cytoplasmic networkingnew connections and more functions, Curr. Opin. Cell Biol, 9, 1997, 49–53.[Medline]
Croop J. & Holtzer H.. Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. I. Light microscope observations, J. Cell Biol, 65, 1975, 271–285.
de Arruda M.V., Watson S., Lin C.S., Leavitt J. & Matsudaira P.. Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins, J. Cell Biol, 111, 1990, 1069–1179.
De Pasquale J.A. & Izzard C.S.. Evidence for an actin-containing cytoplasmic precursor of the focal contact and the timing of incorporation of vinculin at the focal contact, J. Cell Biol, 105, 1987, 2803–2809.
Dubreuil R.R.. Structure and evolution of the actin crosslinking proteins, Bioessays, 13, 1991, 219–226.[Medline]
Geiger B., Volk T., Volberg T. & Bendori R.. Molecular interactions in adherens-type contacts, J. Cell Sci. Suppl, 8, 1987, 251–272.[Medline]
Goldman R.D. & Knipe D.M.. Functions of cytoplasmic fibers in nonmuscle cell motility, Cold Spring Harbor Symp. Quant. Biol., 37, 1972, 523–534.
Goldsmith S.C., Pokala N., Shen W., Fedorov A.A., Matsudaira P. & Almo S.C.. The structure of an actin-crosslinking domain from human fimbrin, Nat. Struct. Biol, 4, 1997, 708–712.[Medline]
Green K.J., Talian J.C. & Goldman R.D.. Relationship between intermediate filaments and microfilaments in cultured fibroblastsevidence for common foci during cell spreading, Cell Motil. Cytoskelet, 6, 1986, 406–418.[Medline]
Hanein D., Matsudaira P. & De Rosier D.J.. Evidence for a conformational change in actin induced by fimbrin (N375) binding, J. Cell Biol, 139, 1997, 387–396.
Hanein D., Volkmann N., Goldsmith S., Michon A.-M., Lehman W., Craig R., DeRosier D., Almo S. & Matsudaira P.. An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation, Nat Struct. Biol., 5, 1998, 23–28.
Hartwig J.H. & Kwiatkowski D.J.. Actin-binding proteins, Curr. Opin. Cell Biol, 3, 1991, 87–97.[Medline]
Herrmann H., Haner M., Brettel M., Muller S.A., Goldie K.N., Fedtke B., Lustig A., Franke W.W. & Aebi U.. Structure and assembly properties of the intermediate filament protein vimentinthe role of its head, rod and tail domains, J. Mol. Biol, 264, 1996, 933–953.[Medline]
Inagaki M., Matsuoka Y., Tsujimura K., Ando S., Tokui T., Takahashi T. & Inagaki N.. Dynamic property of intermediate filamentsregulation by phosphorylation, Bioessays, 18, 1996, 481–487.
Izzard C.S.. A precursor of the focal contact in cultured fibroblasts, Cell Motil. Cytoskelet, 10, 1988, 137–142.[Medline]
Lin C.S., Park T., Chen Z.P. & Leavitt J.. Human plastin genes. Comparative gene structure, chromosome location, and differential expression in normal and neoplastic cells, J. Biol. Chem, 268, 1993, 2781–2792.
Lin C.S., Shen W., Chen Z.P., Tu Y.H. & Matsudaira P.. Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney, Mol. Cell Biol, 14, 1994, 2457–2467.
Liu C.G., Maercker C., Castanon M.J., Hauptmann R. & Wiche G.. Human plectinorganization of the gene, sequence analysis, and chromosome localization (8q24), Proc. Natl. Acad. Sci. USA, 93, 1996, 4278–4283.
Mabuchi K., Li B., Ip W. & Tao T.. Association of calponin with desmin intermediate filaments, J. Biol. Chem, 272, 1997, 22662–22666.
Marchisio P.C., Cirillo D., Teti A., Zambonin-Zallone A. & Tarone G.. Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions, Exp. Cell Res, 169, 1987, 202–214.[Medline]
Matsudaira P.. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J. Biol. Chem, 262, 1987, 10035–10038.
Matsudaira P.. Modular organization of actin crosslinking proteins, Trends Biochem. Sci, 16, 1991, 87–92.[Medline]
Matsudaira P.. Actin crosslinking proteins at the leading edge, Semin. Cell Biol, 5, 1994, 165–174.[Medline]
Matsudaira P.T. & Burgess D.R.. Identification and organization of the components in the isolated microvillus cytoskeleton, J. Cell Biol, 83, 1979, 667–673.
McLean W.H., Pulkkinen L., Smith F.J., Rugg E.L., Lane E.B., Bullrich F., Burgeson R.E., Amano S., Hudson D.L. & Owaribe K.. Loss of plectin causes epidermolysis bullosa with muscular dystrophycDNA cloning and genomic organization, Genes Dev, 10, 1996, 1724–1735.
Merdes A., Brunkener M., Horstmann H. & Georgatos S.D.. Filensina new vimentin-binding, polymerization-competent, and membrane-associated protein of the lens fiber cell, J. Cell Biol, 115, 1991, 397–410.
Messier J.M., Shaw L.M., Chafel M., Matsudaira P. & Mercurio A.M.. Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages, Cell Motil. Cytoskelet, 25, 1993, 223–233.[Medline]
O'Farrell P.Z., Goodman H.M. & O'Farrell P.H.. High resolution two-dimensional electrophoresis of basic as well as acidic proteins, Cell, 12, 1977, 1133–1141.[Medline]
Otto J.J.. Actin-bundling proteins, Curr. Opin. Cell Biol, 6, 1994, 105–109.[Medline]
Perides G., Kuhn S., Scherbarth A. & Traub P.. Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C, Eur. J. Cell Biol, 43, 1987, 450–458.[Medline]
Prahlad V., Yoon M., Moir R.D., Vale R.D. & Goldman R.D.. Rapid movements of vimentin on microtubule trackskinesin-dependent assembly of intermediate filament networks, J. Cell Biol, 143, 1998, 159–170.
Schliwa M., The CytoskeletonAn Introductory Survey, 1986, Springer-Verlag Inc, Viennapp. 326 pp.
Seifert G.J., Lawson D. & Wiche G.. Immunolocalization of the intermediate filament-associated protein plectin at focal contacts and actin stress fibers, Eur. J. Cell Biol, 59, 1992, 138–147.[Medline]
Shevchenko A., Wilm M., Vorm O. & Mann M.. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels, Anal. Chem, 68, 1996, 850–885.[Medline]
Shoeman R.L., Mothes E., Kesselmeier C. & Traub P.. Intermediate filament assembly and stability in vitroeffect and implications of the removal of head and tail domains of vimentin by the human immunodeficiency virus type 1 protease, Cell Biol. Int. Rep, 14, 1990, 583–594.[Medline]
Soellner P., Quinlan R.A. & Franke W.W.. Identification of a distinct soluble subunit of an intermediate filament proteintetrameric vimentin from living cells, Proc. Natl. Acad. Sci. USA, 82, 1985, 7929–7933.
Svitkina T.M., Verkhovsky A.B. & Borisy G.G.. Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton, J. Cell Biol, 135, 1996, 991–1007.
Traub P. & Vorgias C.E.. Involvement of the N-terminal polypeptide of vimentin in the formation of intermediate filaments, J. Cell Sci, 63, 1983, 43–67.[Abstract]
Wang E. & Choppin P.W.. Effect of vanadate on intracellular distribution and function of 10-nm filaments, Proc. Natl. Acad. Sci. USA, 78, 1981, 2363–2367.
Wiche G., Becker B., Luber K., Weitzer G., Castanon M.J., Hauptmann R., Stratowa C. & Stewart M.. Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central -helical coiled coil, J. Cell Biol, 114, 1991, 83–99.
Wiche G., Gromov D., Donovan A., Castanon M.J. & Fuchs E.. Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association, J. Cell Biol, 121, 1993, 607–619.
Yang Y., Dowling J., Yu Q.C., Kouklis P., Cleveland D.W. & Fuchs E.. An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments, Cell, 86, 1996, 655–665.[Medline]
Yoon M., Moir R.D., Prahlad V. & Goldman R.D.. Motile properties of vimentin intermediate filament networks in living cells, J. Cell Biol, 143, 1998, 147–157.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
Related Article
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
