p116Rip Is A Novel Filamentous Actin-binding Protein*

Jacqueline Mulder {ddagger}, Mieke Poland {ddagger}, Martijn F. B. G. Gebbink {ddagger} §, Jero Calafat ¶, Wouter H. Moolenaar {ddagger} || and Onno Kranenburg {ddagger} §

From the {ddagger}Division of Cellular Biochemistry and Centre for Biomedical Genetics, and the Division of Cell Biology, Plesmanlaan 121, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

Received for publication, March 7, 2003 , and in revised form, April 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1–382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (Kd ~ 0.5 µM) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dynamic remodeling of the actin-based cytoskeleton drives cell shape changes, cell division, and motility. Cytoskeletal remodeling involves the assembly and disassembly of filamentous actin (F-actin)1 and is effectuated by cell-surface receptors that signal through small GTPases of the Rho family, notably RhoA, Rac and Cdc42 (1, 2). Many different actin-associated proteins participate in regulating actin dynamics in concert with Rho GTPases (39). Some actin-binding proteins promote organization of actin into higher-order structures, whereas others control actin remodeling in response to extracellular stimuli such as growth factors, hormones, or cell adhesion cues. Although only few proteins bind actin monomers, there are more than 100 that bind polymeric F-actin, and many of them induce cross-linking or bundling of F-actin (10).

In our ongoing studies to delineate Rho signaling by the lipid growth factor lysophosphatidic acid (LPA) (11, 12), we previously identified a ubiquitously expressed protein of 116 kDa, provisionally named p116Rip, which binds relatively weakly to activated RhoA in a yeast two-hybrid assay (13). The p116Rip sequence predicts several protein interaction domains, including at least one PH domain, two proline-rich stretches, and a C-terminal region predicted to form a coiled-coil domain. This suggests that p116Rip may have a scaffolding role recruiting different proteins into a RhoA-regulated macromolecular complex. When overexpressed in N1E-115 neuroblastoma cells, p116Rip promotes cell flattening and process extension and inhibits cytoskeletal contraction in response to LPA (13). The p116Rip phenotype was reminiscent of what is observed after RhoA inactivation (using dominant-negative RhoA or C3 toxin), which led to the suggestion that p116Rip may negatively regulate RhoA signaling (13). However, the function of p116Rip remains unknown; importantly, no evidence that p116Rip binds directly to RhoA in mammalian cells has been discovered (13).

In the present study, we set out to characterize p116Rip in further detail. We find that p116Rip, rather than directly binding to RhoA, interacts with F-actin via its N-terminal region and colocalizes with dynamic F-actin structures such as stress fibers, cortical microfilaments, filopodia, and lamellipodial ruffles. Furthermore, we show that p116Rip induces bundling of F-actin in vitro, with the bundling activity residing in the N-terminal region. Yet overexpression of p116Rip or its N-terminal actin-binding domain disrupts the actin cytoskeleton and thereby interferes with growth factor-induced contractility and lamellipodia formation. Our studies specify p116Rip as a novel F-actin-binding protein and demonstrate that p116Rip can affect, either directly or indirectly, the integrity of the actomyosin-based cytoskeleton.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—N1E-115 and COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 7.5% fetal calf serum. NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. N1E-115 cells were transiently transfected using the calcium phosphate precipitation method described previously (14). NIH3T3 cells were transfected using Lipo-fectAMINE Plus (Invitrogen), and serum was added to the cells after the transfection procedure as described by the manufacturer. COS-7 cells were transfected by the DEAE-dextran method as described previously (15).

Expression Constructs—Generation of full-length (FL) pcDNA3-p116Rip (aa 1–1024), pcDNA3-HA-CTp116Rip (aa 545–1024), pcDNA3-HA-RBDp116Rip (aa 545–823), and prp261-RBDp116Rip (aa 545–823) has been described previously (13). pcDNA3-HA-FLp116Rip was engineered by use of a polylinker created by annealing primers 5'-ggatggcttacccatacgatgttccagattacgcgtgc-3' and 5'-acgcgtaatctggaacatcgtatgggtaagccatccgc-3' encoding the HA-tag sequence and a SacII restriction site. The polylinker was ligated into pcDNA3-FLp116Rip via the SacII site. pcDNA3-HA-NTp116Rip (aa 1–382) and GST-NT were generated by PCR using primers 5'-cggggtaccacatgtcggcggccaaggaa-3' (forward) and 5'-cggaattccggcgtcatggaggattctgt-3' (reverse) for NT. GST-CT was generated from pcDNA3-HA-CTp116Rip. GST-{Delta}5 (aa 1–152), GST-{Delta}6 (aa 1–212), GST-{Delta}7 (aa 43–152), GST-{Delta}8 (aa 43–212); GST-{Delta}9 (aa 212–390) were generated similarly by PCR using specific primers. The PCR products were digested with KpNI-EcoRI and ligated into pcDNA3-HA and pRP265, a derivative of pGEX-1N. FLp116Rip-peGFPN1-was constructed by ligation of a HindIII-ScaI fragment out of pcDNA3-FLp116Rip and a PCR fragment using primers 5'-cagagcagtactcccaaaagtgcctgg-3' (forward) and 5'-cgcggtaccagtcgacagaattcgttatcccatgagac-3' (reverse), encoding ScaI and Asp718 restriction sites, into peGFPN1 (Clontech). pMT2sm-FLp116Rip-GST was generated by ligation of a NotI-ScaI fragment of pcDNA3-FLp116Rip and a PCR fragment using primers 5'-cagagcagtactcccaaaagtgcctgg-3' (forward) and 5'-cggggtaccggaattcgttatcccatgagacctg-3' (reverse), encoding ScaI and Asp718 restriction sites, into pMT2sm-GST. pMT2sm-FLp116Rip-myc was generated by cloning FLp116Rip-myc into pMT2sm. Sequence of all constructs was verified by automated sequencing.

Solubility Assay—Cells were lysed in ice-cold lysis buffer (0.1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, and 1 mM EDTA, supplemented with protease inhibitors) and were left on ice for 15 min. Lysates were centrifuged for 30 min (13,000 rpm; Eppendorf table centrifuge, 4 °C). Pellet and supernatant fractions were collected, dissolved in sample buffer, and subjected to SDS-PAGE. Proteins were detected by Western blotting using the polyclonal anti-p116Rip antibody (1:1000 dilution).

Expression and Purification of Recombinant Fusion Proteins—The E. coli strains BL21 or DH5{alpha} were transformed with plasmids encoding GST-NT, GST-CT, GST-{Delta}5, GST-{Delta}6, GST{Delta}7, GST{Delta}8, GST{Delta}9, or GST, respectively. Colonies were obtained and used to inoculate Luria broth/ampicillin. Cultures were grown and isopropyl {beta}-D-thiogalactoside was added overnight to induce expression of the fusion proteins when the cultures reached an OD between 0.4 and 0.6. Bacteria were harvested by centrifugation at 4000 x g, resuspended in cold lysis buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), and lysed by sonification followed by the addition of 0.5% Nonidet P-40. Lysates were cleared by centrifugation at 4000 x g, and resulting supernatants were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences). Beads with affinity-bound proteins were washed five times with lysis buffer, and bound proteins were eluted with 50 mM Tris-HCl, pH 8.0, containing 10 mM reduced glutathione. In some cases, GST was cleaved off by incubating beads with affinity-bound proteins with thrombin (Amersham Biosciences) according to manufacturer's protocol.

Purified full-length p116Rip fused to GST or purified full-length p116Rip fused to Myc were obtained by transfection of COS-7 cells with the constructs pMT2sm-FLp116Rip-GST and pMT2sm-FLp116Rip-myc, respectively. Cells were lysed in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min). Further purification of the full-length GST fusion proteins occurred in the same manner as the purification of GST fusion proteins produced in bacteria. FLp116Rip-myc fusion protein was purified using a column of monoclonal myc antibodies (9E10) chemically cross-linked to protein G-Sepharose beads (Amersham Biosciences). The protein was eluted off with 0.1 M glycine, pH 2.5, and fractions were collected in tubes containing 0.1 volume of 1 M Tris-HCl, pH 8.0, to make an end pH of 7.0. Eluted proteins were subjected to SDS-PAGE, followed by protein staining with Coomassie Blue to estimate the purity and concentration of the proteins in the fractions. Some of the proteins were concentrated using Centricon 10-kDa cutoff devices (Millipore). Protein concentration was also determined by the Bradford method using BSA as a standard. Purified proteins were stored in aliquots at -80 °C in 10% glycerol.

F-actin Cosedimentation Assay—COS-7 cells were transfected with the indicated expression vectors and lysed for 48 h after transfection in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min) and supernatant aliquots were run out on SDS-PAGE gels to check for expression (data not shown). 10-µl aliquots were used in the in vitro actin-binding assay.

F-actin cosedimentation assays were performed according to the manufacturer's protocol (Cytoskeleton, Denver, CO). Briefly, prespun aliquots of COS-7 cell lysates (100,000 x g, 30 min) or purified proteins GST-NTp116Rip, GST, FLp116Rip-GST, BSA, {alpha}-actinin (Cytoskeleton), or GST-{Delta}5p116Rip; GST-{Delta}6p116Rip; GST-{Delta}7p116Rip; GST-{Delta}8p116Rip; GST-{Delta}9p116Rip were incubated for 1 h at room temperature with 40 µg of pure actin filaments. The final concentration of F-actin was 18 µM. Filaments were subsequently pelleted by centrifugation 100,000 x g (Beckman airfuge). As a control for actin-independent sedimentation, the various proteins were also centrifuged under conditions in which filamentous actin was omitted from the mix. Cosedimenting proteins were resolved by SDS-PAGE and detected by either Coomassie Blue staining or by Western blot analysis using anti-p116Rip antibodies, anti-GFP rabbit polyclonal antibodies (16), or an anti-actin mouse monoclonal antibody (Mab 1501R; Chemicon).

For quantitative analysis, a fixed concentration of FLp116Rip-GST (1 µM) was mixed with increasing amounts of F-actin (0–3.5 µM) in polymerization buffer and incubated at room temperature for 30 min. Proteins were centrifuged as above and total pellets and supernatants were loaded separately on SDS-polyacrylamide gels. Protein bands were detected by Coomassie Blue staining and were scanned and quantified using the software program TINA. The amount of p116Rip bound to different concentrations of F-actin was fit to a single rectangular hyperbola using Prism (ver. 3; GraphPad Software, San Diego, CA). In all cases, entire pellet and supernatant fractions were loaded separately on SDS-polyacrylamide gels and detected by either Coomassie Blue staining or by Western blot (above).

Electron Microscopy—To test for bundling activity, actin filaments (5 µM) were incubated for 30 min with purified proteins GST, {alpha}-actinin (both 2 µM), FL-p116Rip (0.5 µM), NT-p116Rip (0.5 µM), and CT-p116Rip (0.5 µM) at room temperature. Samples were absorbed on to glow-discharged carbon-coated formvar film on a copper grid and negatively stained with 1% uranyl acetate and examined with a Philips CM10 electron microscope.

Metabolic Labeling—N1E-115 cells were incubated in methionine/cysteine-free media for 30 min and labeled for 4 h with medium containing [35S]methionine/cysteine (200 µCi/ml; Amersham Biosciences). Labeled medium was aspirated and cells were washed once in ice-cold PBS. Cells were scraped in ice-cold lysis buffer (1% Nonidet P-40, 50 mM Tris pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, and 10% glycerol, supplemented with protease inhibitors). Lysates were cleared by centrifugation (13,000 rpm; 10 min) and supernatants were tumbled with protein A-Sepharose beads precoupled to either preimmune rabbit serum, anti-p116Rip antibodies (13), or anti-myosin IIA antibodies (BTI, Oklahoma City, OK) for1hat4 °C. GST pull-down assays were performed with 20 µl of GSH-Sepharose beads loaded with 20 µg of either GST alone or the GST-NT-fusion protein containing the isolated actin-binding domain. Beads were washed five times in ice-cold lysis buffer and resolved by SDS-PAGE. Proteins were detected by autoradiography. In some cases the gels were blotted and further analyzed by Western blotting to assess the identity of labeled proteins using the polyclonal anti-p116Rip antibody (1:1000 dilution) and anti-myosin II antibodies (1:500 dilution).

Antibodies and Confocal Microscopy—The FRA58 antibody directed against GST-RBD (amino acids 545–823) has been described previously (13). N1E-115 cells and NIH3T3 cells were grown on gelatin-coated glass coverslips in six-well plates. N1E-115 cells were serum-starved overnight and NIH3T3 cells for 7 h. Cells were fixed 24 h after transfection in 3.7% formaldehyde in PBS for 10 min, permeabilized (0.1% Triton X-100/PBS; 2 min), blocked (1% BSA/PBS; 30 min), and incubated with primary antibodies (FRA58 preimmune serum, polyclonal FRA58 anti-p116Rip, 3F10 anti-HA rat monoclonal antibodies (Roche; 1 h). Subsequently, cells were washed and incubated with secondary antibodies (Goat-anti-rabbit-fluorescein isothiocyanate (DAKO) and Goat-anti-rat-fluorescein isothiocyanate (Rockland); 30 min)) together with rhodamine-conjugated phalloidin (Molecular Probes). Coverslips were mounted in Vectashield and analyzed using a Leica TCS-NT confocal microscope.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We originally isolated p116Rip through its interaction with activated RhoA-V14 in yeast two-hybrid assays in which the RhoA isoprenylation site was mutated to prevent membrane targeting (13); no interaction was found with other small GTPases, notably activated RhoB, RhoE, Rac1, Cdc42, and Ras.2 The interaction between p116Rip and RhoA-V14 was relatively weak, however, and in subsequent studies, we have been unable to confirm that p116Rip interacts with RhoA in mammalian cells (13).2 Furthermore, overexpression of p116Rip in COS-7 cells did not significantly influence the activation state of endogenous RhoA.3 We therefore conclude that p116Rip is unlikely to be a high-affinity binding partner and/or negative regulator of RhoA.

p116Rip Localizes to Dynamic Actin-rich Structures and the Nucleus—As a first step in elucidating the function of p116Rip, we examined its subcellular localization in N1E-115 and NIH3T3 cells using a polyclonal anti-p116Rip antibody raised against the putative "RhoA-binding domain" (RBD; Ref. 13). Cells were simultaneously double-stained with rhodamine-phalloidin to visualize F-actin. In serum-deprived N1E-115 cells, endogenous p116Rip colocalizes with F-actin structures, especially the actin-rich microspikes (Fig. 1, top). After stimulation with LPA, a potent activator of RhoA, N1E-115 cells rapidly round up and neurites retract (17, 18). In those contracted cells, p116Rip is found relocalized to the contractile actomyosin ring at the cell cortex (Fig. 1). In NIH3T3 cells, maintained either in serum-free medium or stimulated with LPA, p116Rip colocalizes with F-actin-rich structures, particularly along stress fibers, at cortical microfilaments, and at the leading edge of lamellipodia (Fig. 1, bottom). Of note, p116Rip staining is also detected in the cytoplasm and the nucleus (Fig. 1, bottom). Specificity of the observed immunostaining was confirmed by using the GST-RBD polypeptide antigen (previously termed {Delta}2; ref. 13), which blocked the p116Rip fluorescence signal. Furthermore, p116Rip transfected into COS-7 or N1E-115 cells showed the same subcellular distribution pattern as endogenous p116Rip: colocalization with F-actin-rich structures as well as nuclear and cytoplasmic staining (Fig. 2B and results not shown). No colocalization with endogenous RhoA was detected in either N1E-115 or NIH3T3 cells (results not shown).



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FIG. 1.
Immunofluorescence analysis of p116Rip localization. Cells were grown on glass coverslips and treated as follows: N1E-115 or NIH3T3 cells were cultured in serum-free medium and were either left untreated or were stimulated with LPA (1 µM) for 10 min to induce RhoA-mediated cytoskeletal contraction and cell rounding (N1E-115 cells) or stress fibers (3T3 cells). NIH3T3 cells were used to visualize lamellipodia and membrane ruffles. Abundant filopodia (microspikes) are observed in serum-starved N1E-115 cells. The distribution of endogenous p116Rip was visualized by immunofluorescence using polyclonal anti-p116Rip antibody (raised against part of the coiled-coil region, aa 545–823 (13)), whereas F-actin was detected by rhodamine-conjugated phalloidin. Merges are shown in green (p116Rip) and red (F-actin). p116Rip is seen to colocalize with filopodia, cortical actin, membrane ruffles, and stress fibers. In addition, p116Rip is found in the nucleus (best visible in the two bottom rows) and the cytoplasm. Scale bars, 10 µm.

 


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FIG. 2.
Association of p116Rip with the actin cytoskeleton. A, expression constructs used for transfection in N1E-115 cells encode FLp116Rip and three deletion mutants encompassing the complete C terminus (construct CT; aa 545–1024) or part of the C terminus (construct RBD; aa 545–823) including the "RhoA-binding domain," as indicated, or p116Rip encompassing the N-terminal PH domain (construct NT; aa 1–382.). P-rich, proline-rich regions. The N-terminal PH domain was not recognized earlier (13). B, immunofluorescence analysis of transfected N1E-115 cells using anti-p116Rip antibody and rhodamine-conjugated phalloidin (red staining only in left). Transfected full-length p116Rip colocalizes with cortical actin, whereas truncation mutants CT and RBD are found in the cytoplasm and in the nucleus. C, solubility of transfected full-length or truncated p116Rip in a buffer containing 0.1% Triton-X-100, as examined by subcellular fractionation into a supernatant (s) and pellet (p) fraction. The p116Rip antibody was used for detection on Western blot. Full-length p116Rip is partially insoluble whereas the truncation mutants CT and RBD are largely soluble. IB, immunoblot. D, N1E-115 cells were transfected with an HA-tagged construct encoding a truncated version of p116Rip encompassing the N-terminal PH domain (construct HA-NT; aa 1–382). Cells were fixed 24 h after start of transfection, and the localization of the expressed NT construct was analyzed by immunofluorescence using anti-HA antibody. F-actin was stained with rhodamine-conjugated phalloidin (red staining). It is seen that the NT protein colocalizes with F-actin.

 

The NT Region of p116Rip Dictates Subcellular Localization—The subcellular localization of p116Rip raises the possibility that p116Rip is an F-actin-binding protein. To test this notion, we examined the intracellular localization of distinct domains of p116Rip and determined their detergent solubility. p116Rip contains several putative protein and phospholipid interaction motifs, including a central PH domain, an N-terminal PH domain (aa 43–152; not noted previously (13)); two proline-rich regions, and a C-terminal coiled-coil region (Fig. 2A). The putative "RhoA-binding domain" (RBD) that was isolated in yeast two-hybrid screens (13) overlaps with the coiled-coil region, as indicated in Fig. 2A.

We generated HA-tagged p116Rip and three truncated versions (HA-tagged) encoding the CT coiled-coil region, the RBD and the NT half (NT-p116Rip; Fig. 2A). The various cDNA constructs were transiently transfected into N1E-115 cells and the subcellular localization and detergent solubility of the resulting proteins were analyzed. Transfected HA-p116Rip, like endogenous p116Rip, localizes to cortical F-actin (and the nucleus; data not shown). In contrast, the p116Rip-CT and RBD polypeptides display nuclear and cytoplasmic localization (Fig. 2B). In keeping with these results, the CT and RBD truncation mutants are largely Triton-soluble, whereas full-length p116Rip (transfected and endogenous) is about 50% insoluble (Fig. 2C and results not shown), consistent with association with the cytoskeleton.

Similar to full-length p116Rip, the N terminus of p116Rip (NT-p116Rip) colocalizes with F-actin and is also detectable in the nucleus (Fig. 2D and results not shown). When the NT-p116Rip-expressing cells were analyzed at >48 h after transfection, however, the F-actin cytoskeleton was largely disrupted (see below). Collectively, these results indicate that the N-terminal part of p116Rip (aa 1–382) determines its subcellular localization.

Binding of p116Rip to F-actin—F-actin associates with the motor protein myosin-II to generate contractile forces in non-muscle cells. In metabolically labeled N1E-115 cells, we found that endogenous p116Rip as well as the purified polypeptide NT-p116Rip (fused to GST) coprecipitated with proteins of 43 and 200 kDa (Fig. 3A, lanes 2 and 4, respectively). Immunoblot analysis confirmed that the 43-kDa protein is actin (not shown), and revealed that the 200 kDa protein represents the heavy-chain of non-muscle myosin-II (Fig. 3B). Although the reciprocal precipitations yielded variable results, our data support the notion that p116Rip associates with actomyosin in vivo.



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FIG. 3.
Association of p116Rip with actomyosin complexes in vivo. A, N1E-115 cells were starved in methionine/cysteine-free medium for 30 min and then labeled for 4 h with [35S]methionine/cysteine. Endogenous p116Rip was immunoprecipitated (IP) using polyclonal anti-p116Rip antibodies (arrow). Normal rabbit serum (NRS) was used as a control for immunoprecipitation (left). GST pull-down assays were performed with either GST alone or the GST fusion protein containing the isolated actin-binding domain (GST-NT-p116 Rip; right lanes). Precipitates were subjected to SDS-PAGE and analyzed by autoradiography. B, Western blot analysis of precipitates and GST pull-down assays using polyclonal anti-p116Rip and anti-myosin-II antibodies, endogenous p116Rip, and myosin-II were immunoprecipitated using polyclonal anti-p116Rip antibodies and polyclonal anti-myosin-II antibodies, respectively (left lanes). Normal rabbit serum (NRS) was used as a control for immunoprecipitation. GST pull-down assays were performed with either GST alone or the GST fusion protein containing the isolated actin-binding domain (GST-NT-p116Rip; right lanes). Myosin coprecipitates in p116Rip immuno-complexes and in the GST-NT-p116Rip pulldown assay.

 

We next investigated the actin-binding properties of NT-p116Rip. As shown in Fig. 4A, NT-p116Rip (fused to GST) cosediments with purified F-actin, as did {alpha}-actinin, whereas GST alone and BSA did not. Fusion proteins containing the C-terminal regions of p116Rip (CT and RBD) failed to cosediment with F-actin (results not shown). It thus seems that the N-terminal region of p116Rip contains an F-actin-binding domain. We next examined the binding affinity of full-length p116Rip for F-actin. Increasing concentrations of purified F-actin (0–3.5 µM) were mixed with a fixed amount of p116Rip (1 µM). After high-speed centrifugation, the amount of p116Rip cosedimenting with F-actin was determined. From the resulting binding curve we estimate that p116Rip binds to F-actin with an apparent dissociation constant (Kd) of about 0.5 µM (Fig. 4B).



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FIG. 4.
Direct binding of p116Rip to F-actin in vitro. A, purified proteins GST-NT, GST alone, {alpha}-actinin, p116Rip-GST, p116Rip-Myc, or BSA were incubated with (+) or without (-) in vitro prepared actin filaments. F-actin was subsequently pelleted by ultracentrifugation. Cosedimentation of the various proteins with F-actin was analyzed by SDS-PAGE followed by Coomassie staining of the gel. GST-NT, p116Rip-GST, p116Rip-Myc, and {alpha}-actinin, but not GST or BSA, cosediment with F-actin (right). None of the proteins tested was pelleted if F-actin was omitted form the reaction mixture (left; not shown for BSA and {alpha}-actinin). S, supernatant fraction; P, pellet fraction. B, direct plot of binding of p116Rip-GST to F-actin. A fixed amount of p116Rip-GST (1 µM) was mixed with various amounts of F-actin (0–3.5 µM), followed by ultracentrifugation. Amounts of the free and bound p116Rip were quantified as described. The percentage of bound p116Rip-GST was plotted against the concentration of F-actin. The curve was obtained by nonlinear fitting to a rectangular hyperbola. The apparent Kd was estimated to be ~0.5 µM. C, diagram of purified recombinant p116Rip proteins that have deletions in the actin-binding domain (NT) ({Delta}5, aa 1–152; {Delta}6; aa 1–212; {Delta}7, aa 43–152; {Delta}8, aa 43–212; {Delta}9, aa 212–390) and that are fused to GST (right). Cosedimentation of the deletion mutants with F-actin was determined by Western blot analysis using a GST antibody. ++, stretch of positive residues, KKKRK, at position 157–161.

 

To define the N-terminal regions mediating F-actin binding in further detail, we made various deletion mutants and determined their F-actin cosedimentation properties, as illustrated in Fig. 4C. Whereas p116Rip has no obvious sequence homology to known actin-binding proteins, potential actin-binding motifs include the N-terminal PH domain (19) as well as a stretch of positively charged residues (KKKRK, aa 157–161) that could interact with the highly anionic actin filament (20). We found that one deletion mutant ({Delta}6; aa 1–212), comprising both the PH domain and the positive stretch, can bind F-actin, whereas the other mutants cannot (Fig. 4C). Thus, the extreme N terminus (aa 1–43), the PH domain, and the adjacent cationic residues are all necessary to mediate F-actin binding. Further definition of the critical actin-binding motif(s) within the N-terminal region must await future studies.

p116Rip Induces Bundling of F-actin in Vitro via its N-terminal Region—We next examined the ability of p116Rip and NT-116Rip to induce actin cross-linking in vitro, using {alpha}-actinin as a positive control. Myc-p116Rip and GST-p116Rip were isolated from transfected COS-7 cells using affinity chromatography, and protein purity was determined by Coomassie Blue staining. Myc-p116Rip, like GST-p116Rip, binds F-actin, as shown by cosedimentation assays using lysates from transfected COS-7 cells (see Fig. 6A, left). Because the dimeric nature of GST could mediate artifactual actin cross-linking by GST-p116Rip, we also used Myc-p116Rip. Purified GST-p116Rip, Myc-p116Rip, {alpha}-actinin, or GST alone were incubated with F-actin, and the samples were subsequently analyzed by electron microscopy. In the absence of p116Rip or in the presence of GST alone, long actin filaments were randomly distributed all over the grid and no organized actin bundles were observed (Fig. 5A). In the presence of either GST-p116Rip or Myc-p116Rip, however, F-actin became organized into thick bundles similar to those formed by the actin-bundling protein {alpha}-actinin (Fig. 5, B, C, and D). The bundles consisted of many actin filaments closely aligned in juxtaposition, with no branching of filaments observed.



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FIG. 6.
Expression of full-length p116Rip or the isolated actin-binding domain induces a dendritic morphology and loss of stress fibers in NIH3T3 cells. A, binding of transfected p116Rip to F-actin. COS-7 cells were transfected with the indicated plasmids. Supernatant aliquots were pelleted in either the presence (center blot) or absence (top blot) of F-actin. Cosedimentation of the proteins with F-actin was tested by SDS-PAGE and Western blot using polyclonal anti-p116Rip and anti-GFP antibodies. Actin was detected by PonceauS staining. Transfected FLp116Rip (GFP fusion, Myc-, or HA-tagged), but not GFP alone, is seen to cosediment with F-actin in vitro. B and C, NIH3T3 cells were transfected with either an HA-tagged p116Rip construct, a p116Rip-GFP fusion construct, or GFP alone (C). Cells were fixed and stained 24 h after transfection. Cells expressing GFP, p116Rip-GFP, or HA-FLp116Rip were visualized by immunofluorescence analysis using either a GFP fluorescence or an anti-HA antibody (left). Center column shows staining with rhodamine-conjugated phalloidin (red in the merged pictures). Note the loss of stress fibers and the dendritic-like morphology of cells that overexpress HA-FLp116Rip or p116Rip-GFP, compared with GFP-expressing control cells. D, quantitative analysis of transfected cells containing stress fibers. At least 100 cells with similar expression levels of p116Rip or control constructs were counted for each experiment. Cells containing four or more stress fibers were scored as "cells with stress fibers." To eliminate observer bias, cells were counted using a "blind" setup. Data are the means (± S.D.) of three independent experiments.

 


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FIG. 5.
p116Rip induces bundles of F-actin. Electron micrographs showing negatively stained preparations of actin filaments incubated with GST (A), bundles formed by incubating F-actin with {alpha}-actinin (B), p116Rip-GST (C), p116Rip-Myc (D), NT-p116Rip (E), and CT-p116Rip (F). Scale bars, 50 nm.

 

We also tested the isolated N-terminal actin-binding domain (NT-p116Rip; aa 1–382) and the C-terminal coiled-coil region (CT-p116Rip; aa 545–1024) for bundling activity. In these experiments, GST-fusion proteins were produced in bacteria followed by GST cleavage. As expected, the NT-p116Rip protein induced actin bundling similar to full-length p116Rip, whereas no actin bundles were observed after incubation of F-actin with the CT polypeptide (Fig. 5, E and F). Thus, p116Rip induces bundling of F-actin in vitro through its N-terminal actin-binding domain.

Expression of p116Rip or the N-Terminal Actin-binding Domain Promotes Stress Fiber Disassembly and Process Outgrowth—We investigated the effects of overexpression of p116Rip and its N-terminal region on cell morphology and cytoskeletal organization in NIH3T3 cells. To this end, we used HA-tagged p116Rip and a p116Rip-GFP fusion protein (its direct binding to F-actin was confirmed; Fig. 6A). Contrary to expectations raised by the actin-bundling studies, overexpression of p116Rip in NIH3T3 cells resulted in loss of stress fibers and outgrowth of long dendrite-like processes (Fig. 6B). This phenotype was observed with wild-type p116Rip, Myc-, HA- and p116Rip-GFP (Fig. 6, B and C, and results not shown). Less than 10% of the p116Rip-transfected NIH3T3 cells contained stress fibers, compared with >60% of the GFP-expressing control cells (Fig. 6D). LPA stimulation of NIH3T3 cells leads to rapid RhoA-mediated cell contraction (albeit less dramatic than in N1E-115 cells). However, no contractile response to LPA was seen in the p116Rip-overexpressing NIH3T3 cells, similar to what we previously observed in p116Rip-overexpressing N1E-115 cells (13). Loss of stress fibers was already detectable at 6 to 8 h after transfection, whereas process extension appeared at later time points (>12–16 h, when p116Rip levels were more elevated).

Expression of the actin-binding region (HA-NT-p116Rip) in NIH3T3 cells led to the same dramatic loss of stress fibers and induction of dendrite-like extensions. In contrast, cells expressing the C-terminal domain only (HA-CT-p116Rip) displayed a normal stress fiber pattern (Fig. 7, A and B). Thus, the NT region of p116Rip is necessary and sufficient for stress-fiber disruption and consequent loss of contractility in p116Rip-overexpressing cells.



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FIG. 7.
Loss of stress fibers induced by p116Rip and its actin-binding domain. A, NIH3T3 cells expressing the deletion constructs were analyzed for the presence of stress fibers. At least 100 cells with similar expression levels of HA-NT-p116Rip or HA-CT-p116Rip were counted for each experiment. Cells containing four or more stress fibers were scored as "cells with stress fibers." To eliminate observer bias, cells were counted by using a blind setup. Data are the means (±S.D.) of three independent experiments. B, NIH3T3 cells were transfected with the indicated deletion mutants, fixed, and immunostained 24 h after transfection. Cells expressing p116Rip deletion mutants were visualized by immunofluorescence using anti-HA antibody (left). Center column shows staining with rhodamine-conjugated phalloidin (red in the merged pictures).

 

Finally, we examined the cytoskeletal response of p116Rip-overexpressing NIH3T3 cells to platelet-derived growth factor (PDGF), which is a potent inducer of Rac-mediated lamellipodia formation and membrane ruffling. PDGF induced prominent lamellipodia formation in the control cells but not in the p116Rip-GFP-expressing cells (Fig. 8, A and B). We conclude that although p116Rip has actin-bundling activity in vitro, overexpression of p116Rip in fibroblasts and neuronal cells disrupts F-actin assembly and thereby interferes with Rho/Rac-controlled cytoskeletal remodeling.



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FIG. 8.
p116Rip overexpression inhibits lamellipodia formation by PDGF. A, NIH3T3 cells were transfected with GFP or p116Rip-GFP constructs (left). Cells were serum-starved for 7 h before stimulation with PDGF (25 ng/ml). Cells were fixed and immunostained 24 h after transfection. Center column shows staining with rhodamine-conjugated phalloidin (red in the merged pictures). B, transfected cells were analyzed for the presence of lamellipodia. At least 100 cells expressing p116Rip or control constructs were counted for each experiment. Data are the means ± S.D. of three independent experiments.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We originally isolated p116Rip as a RhoA-interacting protein in a yeast two-hybrid screen (13). Binding to activated RhoA was relatively weak, however, and our initial conclusion that p116Rip interacts directly with RhoA in mammalian cells turned out to be premature (13). In fact, we have since found that p116Rip is unlikely to be a direct binding partner of RhoA.2 In the present study, we provide the first insights into the function of p116Rip. We show here that p116Rip is an F-actin-binding protein that has bundling activity in vitro, with the actin-binding domain residing in the N-terminal region (aa 1–212; construct {Delta}6-p116Rip; Fig. 4C). This conclusion is based on the following observations: 1) p116Rip and NT-p116Rip associate with actomyosin complexes in vivo; 2), the N-terminal region of p116Rip, but not its C-terminal half, cosediments with F-actin in vitro; and 3), purified full-length p116Rip and NT-p116Rip induce the assembly of actin filaments into thick bundles in vitro. In addition, we show that endogenous p116Rip localizes to dynamic F-actin-rich structures that are normally under the control of Rho family GTPases, notably along stress fibers, in cortical microfilaments as well as in filopodia and lamellipodia. Furthermore, p116Rip is also detected in the nucleus, consistent with p116Rip containing several potential nuclear localization signals (between residues 43–587; not shown). There is growing evidence for the presence of actin and actin-binding proteins in the nucleus, but very little is still known about their importance for normal cell function (21). One challenge for future studies is to determine how nuclear targeting of p116Rip is normally regulated.

The N-terminal region p116Rip shows no obvious sequence similarity to known F-actin-binding proteins. Therefore, p116Rip does not classify as a member of the superfamily of actin-binding proteins that includes {alpha}-actinin/spectrin members, plectin, filamin, and dystrophin (2224). At least three distinct mechanisms could account for the actin-bundling activity of NT-p116Rip. One possibility would be that NT-p116Rip is able to dimerize and thereby induces actin bundling. However, using transfected COS cells, we did not detect an interaction between NT-p116Rip and full-length p116Rip (results not shown), which argues against the possibility that the NT domain can form dimers. The second possibility is that NT-p116Rip might bundle F-actin through the polycationic KKKRK motif (residues 157–161), just after the first PH domain (20). However, mutational analysis reveals that neither the first PH domain nor the cationic motif is sufficient for F-actin binding (Fig. 4C). A third possibility is that NT-p116Rip may harbor two actin-binding domains, each of which binds a separate actin filament; as yet, we have no evidence for or against this notion. Further studies are required to identify the N-terminal sequence motifs in p116Rip that determine F-actin binding and bundling.

Contrary to what one would expect for a protein with actin-bundling activity, overexpression of p116Rip in NIH3T3 cells causes loss of stress fibers and produces a dendrite-like morphology. This phenotype, which is reminiscent of cells expressing dominant-negative RhoA (18, 25) requires the N-terminal actin-binding domain of p116Rip but not the C-terminal coiled-coil region. The importance of the N terminus in determining cytoskeletal architecture can also be inferred from the observation that overexpressed p116Rip causes cell flattening in N1E-115 cells, whereas an N-terminally truncated version does not (13). Loss of stress fibers and other actin-rich structures is a common feature of overexpressed actin-monomer (G-actin) sequestering proteins (2628), but our efforts to test whether NT-p116Rip can bind G-actin yielded negative results (not shown). However, there is precedent for actin cross-linking proteins to cause F-actin disassembly in vivo. In particular, overexpression of the actin-binding region of neurabin, an F-actin cross-linking protein, causes collapse of stress fibers and promotes filopodial outgrowth, apparently by recruiting protein phosphatase I to F-actin-rich structures (29). Furthermore, overexpression of villin, a protein that can bundle, cap, nucleate, or sever actin in vitro, results in the disappearance of stress fibers and enhanced microvilli elongation, a phenotype that strictly correlates with the actin-bundling activity of villin (30).

Overexpressed p116Rip not only induces an inactive RhoA phenotype but also interferes with PDGF-induced lamellipodia formation, which is a typical Rac-mediated response. The present findings lead us to suggest that, rather than being a negative regulator of Rho/Rac, p116Rip can destabilize F-actin-rich structures by competing with and displacing other actin-cross-linking proteins. An alternative or additional possibility is that p116Rip may recruit regulatory proteins that disassemble the F-actin network (such as actin-severing proteins or protein phosphatases; see Ref. 29). As for the displacement model, the neuronal F-actin-binding protein drebrin induces the formation of highly branched processes, similar to that observed with p116Rip. It does so by interfering with the actin binding and bundling activities of fascin, {alpha}-actinin, and tropomyosin (31, 32). A similar mechanism might underlie the p116Rip overexpression phenotype.

Finally, we note that a recently identified actin-binding protein named Tara (593 residues) shows a high degree of similarity to p116Rip (46% overall amino acid identity (33)). In common with p116Rip, Tara contains an N-terminal PH domain and a C-terminal coiled-coil region, but it lacks the N-terminal actin-binding region of p116Rip. No actin cross-linking activity has been reported for Tara until now; nevertheless, overexpression of Tara in HeLa cells leads to enhanced formation of stress fibers and cortical F-actin (33). Thus, despite their structural similarities, p116Rip and Tara have opposing actions on F-actin organization.

In conclusion, our studies specify p116Rip as a novel F-actin-binding protein with bundling activity in vitro and demonstrate that p116Rip can affect, directly or indirectly, the integrity and contractility of the actomyosin-based cytoskeleton. Further insight into the physiological role of p116Rip in cytoskeletal regulation will rely on the identification of additional binding partners of p116Rip as well as on interference approaches by using RNA interference-expressing vectors. These studies are currently in progress.


    FOOTNOTES
 
* This work was supported by the Dutch Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands. Back

|| To whom correspondence should be addressed. Tel.: 31-20-512-1971; Fax: 31-20-512-1989; E-mail: w.moolenaar{at}nki.nl.

1 The abbreviations used are: F-actin, filamentous actin; LPA, lysophosphatidic acid; PH, pleckstrin homology; aa, amino acids; HA, hemagglutinin; FL, full-length; GST, glutathione S-transferase; GFP, green fluorescent protein; NT, N terminus; CT, C terminus; BSA, bovine serum albumin; RBD, RhoA-binding domain; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor. Back

2 J. Mulder, O. Kranenburg, and M. Poland, unpublished results. Back

3 F. van Horck and J. Mulder, unpublished results. Back


    ACKNOWLEDGMENTS
 
We thank Lauran Oomen and Lenny Brocks for assistance with confocal microscopy.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ridley, A. J. (2001) Trends Cell Biol. 11, 471-477[CrossRef][Medline] [Order article via Infotrieve]
  2. Etienne-Manneville, S., and Hall, A. (2002) Nature 420, 629-635[CrossRef][Medline] [Order article via Infotrieve]
  3. Higgs, H. N., and Pollard, T. D. (2001) Annu. Rev. Biochem. 70, 649-676[CrossRef][Medline] [Order article via Infotrieve]
  4. Janmey, P. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14745-14747[Free Full Text]
  5. Borisy, G. G., and Svitkina, T. M. (2000) Curr. Opin. Cell Biol. 12, 104-112[CrossRef][Medline] [Order article via Infotrieve]
  6. Wear, M. A., Schafer, D. A., and Cooper, J. A. (2000) Curr. Biol. 10, R891-R895[CrossRef][Medline] [Order article via Infotrieve]
  7. Svitkina, T. M., and Borisy, G. G. (1999) J. Cell Biol. 145, 1009-1026[Abstract/Free Full Text]
  8. Ridley, A. J. (1999) Nat. Cell Biol. 1, E64-E66[CrossRef][Medline] [Order article via Infotrieve]
  9. Ayscough, K. R. (1998) Curr. Opin. Cell Biol. 10, 102-111[CrossRef][Medline] [Order article via Infotrieve]
  10. dos Remedios, C. G., and Thomas, D. D. (2001) Results Probl. Cell Differ. 32, 1-7[Medline] [Order article via Infotrieve]
  11. Moolenaar, W. H. (1999) Exp. Cell Res. 253, 230-238[CrossRef][Medline] [Order article via Infotrieve]
  12. Kranenburg, O., Poland, M., van Horck, F. P., Drechsel, D., Hall, A., and Moolenaar, W. H. (1999) Mol. Biol. Cell 10, 1851-1857[Abstract/Free Full Text]
  13. Gebbink, M. F., Kranenburg, O., Poland, M., van Horck, F. P., Houssa, B., and Moolenaar, W. H. (1997) J. Cell Biol. 137, 1603-1613; Correction (2001) J. Cell Biol. 153, 133[Abstract/Free Full Text]
  14. Kranenburg, O., Scharnhorst, V., van der Eb, A. J., and Zantema, A. (1995) J. Cell Biol. 131, 227-234[Abstract]
  15. Zondag, G. C., Moolenaar, W. H., and Gebbink, M. F. (1996) J. Cell Biol. 134, 1513-1517[Abstract]
  16. van Ham, S. M., Tjin, E. P., Lillemeier, B. F., Gruneberg, U., van Meijgaarden, K. E., Pastoors, L., Verwoerd, D., Tulp, A., Canas, B., Rahman, D., Ottenhoff, T. H., Pappin, D. J., Trowsdale, J., and Neefjes, J. (1997) Curr. Biol. 7, 950-957[Medline] [Order article via Infotrieve]
  17. Jalink, K., Eichholtz, T., Postma, F. R., van Corven, E. J., and Moolenaar, W. H. (1993) Cell Growth Differ. 4, 247-255[Abstract]
  18. Jalink, K., van Corven, E. J., Hengeveld, T., Morii, N., Narumiya, S., and Moolenaar, W. H. (1994) J. Cell Biol. 126, 801-810[Abstract]
  19. Yao, L., Janmey, P., Frigeri, L. G., Han, W., Fujita, J., Kawakami, Y., Apgar, J. R., and Kawakami, T. (1999) J. Biol. Chem. 274, 19752-19761[Abstract/Free Full Text]
  20. Tang, J. X., and Janmey, P. A. (1996) J. Biol. Chem. 271, 8556-8563[Abstract/Free Full Text]
  21. Rando, O. J., Zhao, K., and Crabtree, G. R. (2000) Trends Cell Biol. 10, 92-97[CrossRef][Medline] [Order article via Infotrieve]
  22. Lappalainen, P., Kessels, M. M., Cope, M. J., and Drubin, D. G. (1998) Mol. Biol. Cell 9, 1951-1959[Free Full Text]
  23. Van Troys, M., Vandekerckhove, J., and Ampe, C. (1999) Biochim. Biophys. Acta 1448, 323-348[Medline] [Order article via Infotrieve]
  24. McCann, R. O., and Craig, S. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5679-5684[Abstract/Free Full Text]
  25. Brouns, M. R., Matheson, S. F., and Settleman, J. (2001) Nat. Cell Biol. 3, 361-367[CrossRef][Medline] [Order article via Infotrieve]
  26. Lee, S. H., Zhang, W., Choi, J. J., Cho, Y. S., Oh, S. H., Kim, J. W., Hu, L., Xu, J., Liu, J., Lee, J. H., and Lee, S. H. (2001) Oncogene 20, 6700-6706[CrossRef][Medline] [Order article via Infotrieve]
  27. Mattila, P. K., Salminen, M., Yamashiro, T., and Lappalainen, P. (2003) J. Biol. Chem. 278, 8452-8459[Abstract/Free Full Text]
  28. Vartiainen, M., Ojala, P. J., Auvinen, P., Peranen, J., and Lappalainen, P. (2000) Mol. Cell. Biol. 20, 1772-1783[Abstract/Free Full Text]
  29. Oliver, C. J., Terry-Lorenzo, R. T., Elliott, E., Bloomer, W. A., Li, S., Brautigan, D. L., Colbran, R. J., and Shenolikar, S. (2002) Mol. Cell. Biol. 22, 4690-4701[Abstract/Free Full Text]
  30. Friederich, E., Vancompernolle, K., Louvard, D., and Vandekerckhove, J. (1999) J. Biol. Chem. 274, 26751-26760[Abstract/Free Full Text]
  31. Ishikawa, R., Hayashi, K., Shirao, T., Xue, Y., Takagi, T., Sasaki, Y., and Kohama, K. (1994) J. Biol. Chem. 269, 29928-29933[Abstract/Free Full Text]
  32. Sasaki, Y., Hayashi, K., Shirao, T., Ishikawa, R., and Kohama, K. (1996) J. Neurochem. 66, 980-988[Medline] [Order article via Infotrieve]
  33. Seipel, K., O'Brien, S. P., Iannotti, E., Medley, Q. G., and Streuli, M. (2001) J. Cell Sci. 114, 389-399[Abstract/Free Full Text]