Correspondence to: Suresh K. Alahari, CB# 7365, ME Jones Bldg., Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599. Tel:(919) 966-4343 Fax:(919) 966-5640 E-mail:alahari{at}med.unc.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrins have been implicated in key cellular functions, including cytoskeletal organization, motility, growth, survival, and control of gene expression. The plethora of integrin and ß subunits suggests that individual integrins have unique biological roles, implying specific molecular connections between integrins and intracellular signaling or regulatory pathways. Here, we have used a yeast two-hybrid screen to identify a novel protein, termed Nischarin, that binds preferentially to the cytoplasmic domain of the integrin
5 subunit, inhibits cell motility, and alters actin filament organization. Nischarin is primarily a cytosolic protein, but clearly associates with
5ß1, as demonstrated by coimmunoprecipitation. Overexpression of Nischarin markedly reduces
5ß1-dependent cell migration in several cell types. Rat embryo fibroblasts transfected with Nischarin constructs have "basket-like" networks of peripheral actin filaments, rather than typical stress fibers. These observations suggest that Nischarin might affect signaling to the cytoskeleton regulated by Rho-family GTPases. In support of this, Nischarin expression reverses the effect of Rac on lamellipodia formation and selectively inhibits Rac-mediated activation of the c-fos promoter. Thus, Nischarin may play a negative role in cell migration by antagonizing the actions of Rac on cytoskeletal organization and cell movement.
Key Words: integrin, Rac, cell migration, cytoskeleton, two hybrid
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The integrin family of cell surface glycoproteins plays a major role in the interaction of cells with the extracellular matrix (/ß heterodimers and each subunit has a large extracellular domain, a single helical transmembrane domain, and, typically, a relatively short cytoplasmic domain. At specialized sites of cellmatrix adhesion, termed focal contacts, integrin cytoplasmic domains articulate, directly or indirectly, with various proteins, including talin,
-actinin, vinculin, paxillin, tensin, and focal adhesion kinase (FAK),1 that are involved in coupling between integrins and the actin cytokeleton (
The cytoplasmic domains of integrins play a key role in their function. Thus, the ß chain cytoplasmic tail has been implicated in the recruitment of integrins to focal contacts ( subunit cytoplasmic tail has been implicated in regulation of integrin affinity (
Integrins can interact with a variety of partner proteins, including various membrane receptors that bind to the extracellular and transmembrane domains of integrins ( chain cytoplasmic domains. Thus, calreticulin has been reported to bind the conserved GFFKR motif found in all
chains and to modulate integrin affinity (
IIb, possibly playing a role in activation of the
IIbß3 integrin (
The 5ß1 integrin, a receptor for fibronectin, seems to play a special role in regulating growth and survival in some cell types. Thus, high expression of
5ß1 has been linked with reductions in tumor cell growth rates both in vitro and in vivo (
5ß1 also plays a unique role in protecting cells against apoptosis triggered by mitogen deprivation (
5ß1 has been reported to regulate muscle cell growth and differentiation (
5ß1 on growth or apoptosis may be
5 specific, and thus, there may be intracellular proteins that selectively interact with the
5 cytoplasmic tail to mediate these events. Accordingly, we have made use of the yeast two-hybrid system to identify proteins that bind to the
5 cytoplasmic domain. We have identified a novel protein that associates with the cytoplasmic tail of the
5 subunit, and, to a minor degree, with cytoplasmic domains of other
subunits, and that strongly affects cell migration and influences cytoskeletal organization. We named this novel protein Nischarin, which is derived from a term in classic Sanskrit that connotes slowness of motion. This designation is based on the finding, shown below, that overexpression of Nischarin dramatically impairs cell migration.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Two-Hybrid Screening
Here, L40 (Mata his3200 trp1-901, 112 ade2 LYS2::(lexAop)4-HIS3 URA3(lexAop)8-LacZ Gal4) and AMR70 (Mata his3 lys2 trp1 leu2 URA3::(lexAop)8-LacZ Gal4) yeast strains were used (gifts from Dr. Stan Hollenberg, Vollum Institute, Oregon Health Sciences University, Portland, OR). Yeast two-hybrid screening was conducted as previously described (
5 plasmid, which has the
5 cytoplasmic domain fused to the LexA DNA-binding domain and with a tryptophan marker, was transformed into yeast strain L40 and selected for tryptophan prototrophy. Plasmids (pVp16) containing mouse embryonic cDNA libraries of 9.5 and 10.5 d fused to the VP16-transactivating domain and a leucine marker were transformed into the L40 strain containing the bait plasmid and screened for leucine, tryptophan, and histidine prototrophy. A total of 1.7 x 107 transformants were screened for positives. The libraries and vectors were gifts from Dr. Stan Hollenberg. Histidine-positive colonies were further tested for LacZ activation. Dual positives were further confirmed for specificity of the interaction using various baits and included integrin ß1,
2, and
v cytoplasmic domains, as well as lamin, an irrelevant protein in this context. Specificity of the interaction was confirmed by mating experiments. The pBTM bait plasmids were "cured" from dual-positive clones by growth in nonselective medium. The presence of the library plasmids with inserts in the "cured" clones was confirmed by PCR using vector-specific primers. AMR 70 strain cells were transformed separately with pBTM
5, pBTM ß1, pBTM
v, pBTM
2, pBTM lamin, or pBTM vector alone. These transformed cells were mated with the "cured" L40 cells that contained positive pVP16 library plasmids.
Cloning of Full-Length Nischarin
To clone full-length Nischarin, we screened a mouse brain library in the lambda Zap II vector (Stratagene). Using a colony hybridization technique, 30,000 plaques were screened with a 32P-labeled PCR product consisting of 0.45 kb of the integrin-binding region of Nischarin. From this screen, one strong positive plaque was identified and confirmed in two further rounds of screening. Sequence analysis of this clone (A3.1) indicated that the sequence was incomplete at the 3' end. Using a different PCR probe, the lambda Zap library was screened again to obtain the remainder of the Nischarin cDNA. This screen gave several positives, and the longest clone (clone 14.2) was picked. Clones A3.1 and 14.2 provided the complete open reading frame (ORF) of Nischarin.
DNA Constructions and Transfection
The construction of two-hybrid bait plasmids, GST chimeras, and partial- and full-length myc-tagged Nischarin mammalian expression constructs followed standard recombinant DNA procedures. Clones A3.1 and 14.2, mentioned above, were used to make full-length expression constructs. Full details are available upon request. A chimera comprised of full-length Nischarin and GFP was prepared by an inframe insertion of the coding region of Nischarin into the pE-GFP-N1 vector (CLONTECH Laboratories, Inc.). Expression plasmids for CD4, human 2, and
v integrin subunits were obtained from Drs. R. Nicholas (University of North Carolina-Chapel Hill, Chapel Hill, NC), L. Parise (University of North Carolina-Chapel Hill), and David Cheresh (The Scripps Research Institute, La Jolla, CA), respectively. Transfection of mammalian cell lines was usually done with Lipofectamine (GIBCO BRL) or Superfect (QIAGEN), according to the manufacturer's specifications.
Northern Blot Analyses
A mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc.) was probed with a 0.45-kb fragment of the 5 integrinbinding region of Nischarin (probe 1) (nucleotides 1,3051,743), with fragments from the far 5' end (probe 2) (nucleotides -334+56], or the 3' end (probe 3)(nucleotides 2,9363,748). RNA was isolated from various cell lines, run on agarose-formaldehyde gels, and hybridized with probes 13, using previously described techniques (
Antibodies
The predicted ORF of Nischarin was used to design two peptides represented at the far COOH terminus of the protein. The peptides (EALCGRELPVELTGA-C and LDDGRRVRDLDRVL-C) were obtained from the University of North Carolina-Protein Core Laboratory. Both peptides were conjugated to keyhole limpet hemocyanin (Pierce Chemical Co.) and sent to Aves Laboratories for production of chicken pAbs. Anti-5 cytoplasmic domain pAb was a gift from Richard Hynes (Massachusetts Institute of Technology, Cambridge, MA). Anti-myc mAbs and pAbs were purchased from Babco. pAbs to
v cytoplasmic domain were provided by Guido Tarone (University of Torino, Torino, Italy). Rat antimouse
5 mAb, and control rat IgG were purchased from PharMingen and Sigma-Aldrich, respectively. mAbs to vinculin and phosphotyrosine were purchased from Sigma-Aldrich and Upstate Biotechnology. Fluorescent phalloidin was bought from Sigma-Aldrich. A partially purified preparation of the human
5ß1 integrin (Chemicon) was sometimes used as a control.
Binding To GST Fusion Proteins
GSTNischarin fusion proteins expressed from pGEX vectors (Amersham Pharmacia Biotech) were prepared in a standard manner and bound to glutathione-Sepharose 4B beads for "pull down" experiments. CHO cells (clone B227), which overexpress the human
5 integrin subunit, were used as the source of integrins (
5-deficient cells (CHO B2) were used as controls. CHO cells were lysed in a buffer containing nonionic detergent and protease inhibitors. The CHO lysate was added to the GST proteincontaining beads, incubated for 1 h at 4°C, and washed four times with buffer. Bound CHO proteins were eluted by boiling in 2x SDS sample buffer and analyzed by Western blotting using anti-
5 cytoplasmic domain antibody.
Coimmunoprecipitation Experiments
CHO B227 and B2 cells were transiently transfected with myc vector, myc-Nischarin (434581), or myc-Raf. After 48 h of transfection, cells were lysed in a 0.5% Triton X-100 buffer. These lysates were immunoprecipitated with anti-myc antibody, resolved by 7% SDS-PAGE, electrophoretically transferred to nylon membranes, and Western blotted with anti-
5 cytoplasmic domain antibody. In further studies with full-length Nischarin, cells were lysed in a buffer containing 0.1% Triton X-100 (
5,
v, or CD4, were immunoprecipitated with anti-myc and blotted with anti-
5 extracellular domain antibody (Transduction Laboratories), anti-
v, or anti-CD4 antibodies (Santa Cruz Biotechnology, Inc.). For mouse NB41A3 cells, endogenous
5 was immunoprecipitated with rat anti-
5 mAb and the immunoprecipitate was blotted for endogenous Nischarin using the chicken anti-Nischarin pAb described above.
Cell Migration Experiments
Wound-type cell migration experiments were performed as described previously (
Cell migration studies using Nischarin-transfected or control-transfected 3T3 cells or CHO cells were also performed using a transwell assay, according to a previously described procedure (5 or
2 subunits (
5, as well as CHO B2a27, its
5 transfectant (
Subcellular Fractionation
Cos7 cells transfected with myc-Nischarin and untransfected Neuro 2A cells were subjected to subcellular fractionation, as described previously (
Fluorescence Microscopy
Immunofluorescence studies with antibodies to integrins or focal contact proteins were conducted according to procedures described previously (
In some cases, the subcellular distribution of Nischarin was evaluated using the full-length NischarinGFP chimera described above. This was transfected into 3T3 cells at a level of 2 µg per well (it should be noted that levels of expression of NischarinGFP chimeric protein were substantially lower than expression of myc-Nischarin protein when equivalent amounts of plasmid were transfected). After 48 h of transfection, cells were plated onto fibronectin coverslips, as described above, and incubated with antibodies to vinculin or integrins, and then with TRITC-conjugated secondary antibody, or with TRITC-phalloidin to visualize actin. In all cases, coverslips were observed on a ZEISS Axioscop fluorescence microscope using a 40x oil immersion objective. Images were recorded using a CCD camera and a computer with Metamorph image analysis software.
Rho GTPase Experiments
For studies on Rho-mediated signaling, NIH 3T3 cells were cotransfected with 1 µg of luciferase reporter under the control of the c-fos promoter (c-fosLuc) (
To study the effect of Rho-family GTPase on the cytoskeleton, 3T3 cells were transfected with a plasmid expressing an activated (Q61L) form of Rac and with a Nischarin plasmid or with a vector control. A small amount of a GFP-expressing plasmid was used to mark the transfectants. After 48 h, the actin filaments were stained with TRITC-phalloidin and the cells were observed by fluorescence microscopy, as described above.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of a Novel 5-interacting Protein
We used a yeast two-hybrid screen to identify proteins that interact with the 5 cytoplasmic domain. A protein composed of the complete cytoplasmic tail of
5 fused with the DNA-binding domain of Lex A was expressed from the yeast plasmid pBTM
5. We searched mouse embryonic libraries for proteins that interact with the
5 cytoplasmic tail. Protein domains from the libraries were fused to the VP16-transactivating domain and expressed from the yeast plasmid pVP16. Cotransformants of pBTM
5 and pVP16 in the yeast L40 were screened for conversion to histidine prototrophy. Out of 1.7 x 107 transformants screened, 120 colonies were positive for histidine; of those, 45 were also positive for LacZ activation. To determine specificity, several other "baits" were tested for interaction with the
5-binding library protein(s). In particular, we tested for interactions with the cytoplasmic domains of the
2,
v, or ß1 integrin subunits, or with the irrelevant protein lamin. As seen in Fig 1 and Table 1, the
5 bait strongly interacted with the library protein and activated expression of the histidine and LacZ markers. The
v and
2 baits only weakly activated the histidine reporter, and were unable to activate LacZ. These data suggest that the
5-positive library protein may interact weakly with several integrin
subunits, but binds strongly to the
5 subunit.
|
|
Restriction enzyme analysis and DNA sequencing revealed that the insert in all positives tested was comprised of the identical 450 nucleotide sequence. This sequence comprises an ORF, but no start or stop codons. To find the full-length sequence of this cDNA, we screened a mouse brain library in the lambda zap II vector. We identified two overlapping cDNA fragments; and sequence analysis of these fragments revealed the complete ORF for this gene. This consists of 4,062 nucleotides and codes for a novel protein of 1,354 amino acids, with a predicted molecular weight of 148,053. As mentioned above, we have termed this protein Nischarin, a name which describes its effects on cell migration. The predicted amino acid sequence of Nischarin is shown in Fig 2. The validity of this ORF as a protein-coding region is suggested by the presence of a well conserved Kozak sequence immediately 5' of the ATG, the presence of a poly A tail in the 3' untranslated region, the fact that the observed message size (5.5 kb, see below) is consistent with the cDNA size, and the fact that several mouse expressed sequence tags, which have the correct reading frame, overlap our predicted ORF region.
|
Homologies with Other Proteins
BLAST analysis indicates that Nischarin lacks significant homology with any known protein with a well-described function. A close human homologue of Nischarin protein has been reported in Genbank (sequence data available from GenBank/EMBL/DDBJ under accession no. AF082516). It has been suggested, based on limited evidence (
Nischarin Binds Integrin 5 Subunit In Vitro and In Vivo
To confirm that the interaction between the 5 cytoplasmic tail and Nischarin detected by two-hybrid analysis also occurs in vitro, two GSTNischarin constructs were made, GSTNisch (435582) and GSTNisch (33588). GSTNisch (435582) corresponds to the integrin-binding region of Nischarin identified in the two-hybrid system, whereas the second construct contains additional NH2-terminal residues. The NischarinGST fusion proteins were immobilized on a glutathione-agarose matrix and incubated with purified
5ß1 protein, with a cell lysate of CHO B2
27 (human
5transfected cells) or CHO B2 (
5-deficient cells). The proteins retained on the glutathione matrix were analyzed by SDS-PAGE and immunoblotting for
5. Consistent with the yeast data, the GSTNischarin fusion proteins, but not GST alone, were able to interact with purified
5ß1 or with
5ß1 from a cell lysate (Fig 3 A). These data indicate Nischarin binds the
5 integrin subunit in vitro.
|
Several types of coimmunoprecipitation experiments were performed to confirm that Nischarin interacts selectively with the 5 integrin subunit in mammalian cells. First, CHO B2
27 cells were transiently transfected with a construct expressing a truncated myc epitopetagged segment of Nischarin (435582), with myc-tagged Raf, or with myc vector alone. Cells were lysed in a buffer containing nonionic detergent, immunoprecipitated with anti-myc antibody, and blotted with anti-
5 or anti-myc antibodies. Western blotting with anti-
5 antibody indicated that
5 coimmunoprecipitated with anti-myc antibody only in cells transfected with myc-Nischarin (435582), but not in cells transfected with myc-Raf or myc vector alone (Fig 3 B). Expression of similar amounts of myc-Nischarin and myc-Raf was confirmed by Western blotting (data not shown). In experiments with CHO B2 cells, which lack
5, Nischarin did not coimmunoprecipitate a band in the
5 region. This indicates that myc-tagged Nischarin (435582), but not an irrelevant myc-tagged protein, can bind
5ß1 integrin.
Further coimmunoprecipitation experiments were done using full-length Nischarin. To show that Nischarin interacts preferentially with 5 integrins and not with other transmembrane proteins, coimmunoprecipitations were done with CD4, another protein having a single transmembrane domain. Cos 7 cells were transiently cotransfected with myc-Nischarin and pcDNA-
5 or myc-Nischarin and pcDNA-CD4. These cells were lysed and myc immunoprecipitates were processed for blotting with anti-
5 or anti-CD4 antibodies. As shown in Fig 3 C, myc-Nischarin specifically immunoprecipitated
5, but not CD4.
To further examine the alpha subunit selectivity of Nischarin, Cos 7 cells were cotransfected with myc-Nischarin and plasmids expressing the human 5 or
v subunits. These cells were lysed and myc immunoprecipitates were processed for blotting with anti-
5 or anti-
v antibodies. As seen in Fig 3 D, Nischarin coimmunoprecipitated substantial amounts of
5, but barely detectable
v subunits. Thus, the strong preference of Nischarin for the
5 subunit, which was detected in the two-hybrid analysis, seems to be borne out in the cellular setting.
Coimmunoprecipitation experiments were also done for endogenous Nischarin and endogenous 5 subunit. NB41A3 cells, a mouse neuronal-derived line, were lysed and immunoprecipitates formed using rat antimouse
5 mAb, or rat IgG as a control. The immunoprecipitates were Western blotted using chicken anti-Nischarin pAb. As seen in Fig 3 E, a band for Nischarin was detected in the
5 immunoprecipitate, but not in the control. It was not possible to examine other integrin
subunit imunoprecipitates, since only low levels of expression were found in NB41A3 cells for other integrins for which antimouse mAb are available.
The experiments shown in Fig 3 AE indicate: (a) full-length Nischarin or truncated Nischarin (435582) can bind to the 5 integrin subunit, (b) other proteins (e.g., myc-Raf) do not bind
5 under the conditions used for immunoprecipitation, and (c) full-length Nischarin does not bind to coexpressed irrelevant proteins. Furthermore, Nischarin seems to prefer the
5 subunit compared with other tested
subunits. These findings demonstrate a selective interaction between Nischarin and the
5 integrin subunit in mammalian cells. This interaction is able to occur under physiological conditions, as indicated by the coimmunoprecipitation of endogenous
5 and Nischarin.
Tissue Distribution of Nischarin
To determine the tissue distribution of Nischarin mRNA, a mouse multiple tissue Northern blot was hybridized with a PCR probe from the Nischarin integrin-binding region. A single mRNA of 5.5 kb was detected. In further analysis (not shown), three probes from different regions of the cDNA (extreme 5' end, integrin-binding region, and extreme 3' end of the ORF) were used; all three probes detected the same message. Nischarin mRNA expression was highest in brain and kidney, expression levels were lower in heart, liver, lung, and skeletal muscle, whereas no expression was seen in spleen and testis (Fig 4 A). To address the expression of Nischarin in various cell types, we performed Northern blot analysis on several cell lines. Nischarin message was present in various rodent epithelial, fibroblast, and neuronal cell lines, though higher levels tended to be present in neuronal cells (Fig 4 B and data not shown). Analysis of a mouse embryo RNA blot indicated the presence of Nischarin message as early as 7 d of development (Fig 4 C).
|
Expression of Nischarin in Cells and Its Subcellular Localization
The expression of transfected myc-tagged full-length Nischarin and expression of endogenous Nischarin were evaluated by Western blotting of cell lysates with anti-myc antibodies, as well as chicken pAbs directed against polypeptides from the COOH-terminal region of the predicted Nischarin ORF. In Cos7 cells transfected with myc-tagged full-length Nischarin, both antibodies recognized the same band of 190 kD (Fig 5A and Fig B); this is somewhat larger than the predicted molecular weight of expressed Nischarin, but the reason for this is unclear. The expression of endogenous Nischarin was evaluated in detergent lysates of several cell lines by Western blotting with chicken anti-Nischarin antibodies (Fig 5 A). In rat intestinal epithelial (RIE) cells (
190 kD was detected; this band comigrated with the immunoreactive band in Nischarin-transfected Cos7 cells. In NIH 3T3 cells, the chicken anti-Nischarin antibody detected a very weak band of 190 kD (data not shown). In several mouse neuronal cell lines (NIE 119, NB41A3, BC(3)H1, and Neuro 2A), an immunoreactive band of somewhat higher apparent molecular weight was detected (Fig 5 A, and data not shown). The immunoreactivities of the bands detected in Nischarin-transfected Cos7 cells, or in nontransfected RIE cells, were competed out upon addition of the Nischarin COOH-region peptide used for chicken immunization (Fig 5 C). Thus, the bands detected by the chicken antibody in the various rodent cell lines or in the transfected Cos7 cells are very likely to represent Nischarin. The detection of immunoreactive bands of differing apparent molecular weights in the various cell types examined suggests differences in splicing or posttranslational modifications. The fact that only a single Nischarin message is seen by Northern analysis seems to militate against the possibility of splicing differences. However, alternate splicing of a short region of the mRNA might not be readily detected. The data of Fig 5AC, indicate that Nischarin is widely expressed in various rodent cell lines, with higher levels found in cells of neuronal origin.
|
Both biochemical and fluorescence microscopy techniques were used to evaluate the subcellular localization of Nischarin. Overall distribution of Nischarin was assayed by subcellular fractionation. Cos7 cells were transfected with plasmids expressing myc-tagged full-length Nischarin. These cells, as well as untransfected Neuro 2A cells, were fractionated as described in Materials and Methods. Fractions containing membranes or 100,000 g supernatant were prepared, resolved by SDS-PAGE, and subjected to Western blotting with chicken anti-Nischarin antibodies (Fig 6 A). Both transfected and endogenously expressed Nischarin were primarily found in the 100,000 g supernatant fraction, indicating that Nischarin is a soluble rather than a transmembrane protein.
|
We have used the NischarinGFP chimera described in Materials and Methods and fluorescence microscopy to obtain further information on the subcellular localization of Nischarin (Fig 6 B). NIH 3T3 cells expressing the NischarinGFP chimera were counterstained with antibodies to the focal contact protein vinculin (Fig 6 B). NischarinGFP did not enter the nucleus and was primarily found diffusely distributed in the cytosol. However, a greater concentration of Nischarin was seen in the perinuclear region partially associated with punctate structures that may be endomembrane vesicles. There was no evidence of NischarinGFP accumulation at vinculin-rich focal adhesion sites. Staining of focal contacts and fibrillar structures with anti-5 antibody also failed to reveal any obvious colocalization with Nischarin (not shown). Similar studies using an antiphosphotyrosine antibody to detect focal contact sites yielded the same result (data not shown). Thus, Nischarin is found primarily in the cytosol, with some concentration in the perinuclear area, but does not concentrate at "classical" focal adhesion sites.
The observations in Fig 6 indicate that Nischarin is largely a cytosolic protein, and its overall distribution does not coincide with focal adhesion structures. Since Nischarin can clearly associate with the 5 subunit, as demonstrated by coimmunoprecipitation, this may indicate that only a small fraction of the total cellular pool of Nischarin is associated with the
5ß1 integrin at any given time.
Nischarin Inhibits Cell Migration
To investigate the biological role(s) of Nischarin, we focused on the finding that Nischarin seems to interact most strongly with the 5 subunit and on the knowledge that
5ß1 plays an important role in cell motility. The effect of Nicharin on cell migration was initially evaluated using a monolayer "wounding" assay (
|
Nischarin effects on cell movement were also evaluated using a transwell assay (5ß1 expression (
5ß1 and do not adhere to fibronectin, but express other integrins that allow adhesion and migration on vitronectin or other matrix proteins. CHO B2a27 cells derive from B2, but have been stably transfected with human
5; these cells migrate on fibronectin in a completely
5ß1-dependent manner. The transwell membranes for these assays were coated with either fibronectin or vitronectin. As seen in Fig 7 B, transfection of B2a27 cells with Nischarin led to a major reduction of their migration on fibronectin-coated membranes, but only a modest reduction on vitronectin-coated membranes. Furthermore, migration of the B2 cells on vitronectin-coated membranes was not at all affected by expression of Nischarin. Treatment of cells with cytochalasin D completely abolished migration of either cell type.
In another set of experiments, transwell migration assays were performed with 3T3 cells stably transfected with human 5 or
2 subunits (
5-overexpressing cells on fibronectin, but had little effect on the migration of the
2-overexpressing cells on collagen.
Thus, Nischarin overexpression can profoundly inhibit cell migration. This effect displays substantial integrin subunit specificity and is much more dramatic for migration on fibronectin mediated by 5ß1 than for migration on other matrix proteins mediated by other integrins.
Effects of Nischarin on the Cytoskeleton
Since overexpression of Nischarin resulted in substantial alterations in cell migration, we wished to determine if this was accompanied by changes in the organization of the cytoskeleton. REF cells were transfected with plasmids expressing full-length Nischarin and GFP. Cells were probed with the actin-binding reagent phalloidin or were immunostained for phosphotyrosine, vinculin, tubulin, or vimentin. Phalloidin staining (Fig 8 A) indicated that many of the transfected REF cells had a unique phenotype, with a more or less circular shape and having actin filaments arranged in "basket" structures around the periphery, rather than as the linear stress fibers commonly seen in adherent fibroblasts. Although this phenotype was not universal, 60% of the REF cells cotransfected with Nischarin and GFP showed the basket-like actin structures. In contrast, only a few percent of the control GFP transfectants had this phenotype. We next looked for the effects of Nischarin on focal adhesions by staining for vinculin and phosphotyrosine. As seen in Fig 8 B, vinculin-containing focal contacts and phosphotyrosine in focal contacts were somewhat reduced in REF cells transfected with Nischarin compared with control cells. Staining, with anti-tubulin or anti-vimentin antibodies, suggested that Nischarin expression had little effect on the organization of microtubules or intermediate filaments in REF cells (not shown). Similar effects were observed in WI-38 cells, another well spread cell line (not shown). However, the "basket" phenotype was not apparent in Nischarin-transfected NIH 3T3 cells or in Cos7 cells, both of which are less well spread. The highly organized actin filaments of REFs may allow easier visualization of the relatively subtle effects of Nischarin on cytoskeletal architecture.
|
Effects of Nischarin on Rac GTPase-mediated Signaling
Since the organization of the actin cytoskeleton, as well as cell motility, are strongly influenced by the activity of the Rac GTPase (
|
To further evaluate the possible interplay between Nischarin and the Rac GTPase, we examined the effect on Nischarin on a characteristic cytoskeletal function of Rac, namely the enhanced formation of lamellipodia (3540% of the cells displaying large lamellipodia when higher doses of Nischarin were transfected (Fig 10 C). Some of the Rac plus Nischarintransfected cells resembled untransfected 3T3 cells (Fig 10 B). However, various cells at each dose of Nischarin displayed intermediate phenotypes with partial ruffling and incomplete lamellipodia (not shown). Thus, overexpression of Nischarin can inhibit lamellipodia formation, one of the most characteristic effects of Rac on the cytoskeleton and associated with cell movement. These findings suggest that Nischarin inhibits cell migration, at least in part, through its actions on pathways regulated by the Rac GTPase.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies suggest that individual integrin /ß heterodimers can play unique roles in the regulation of cell migration, growth, survival, and differentiation (
5ß1 integrin is particularly interesting in this regard, since it has been implicated in the control of both cell growth and programmed cell death (
5 subunit. Using both two-hybrid analysis and coimmunoprecipitation of expressed proteins in cells, Nischarin was found to interact with the
5 subunit much more strongly than with the other two
subunits tested. Furthermore, immunoprecipitation of endogenous
5ß1 from a mouse neuronal cell line resulted in coimmunoprecipitation of endogenous Nischarin. Thus, current evidence suggests that Nischarin interacts preferentially with the
5 subunit, and this interaction can occur under physiological conditions. However, we cannot rule out the possibility that Nischarin may interact with other examples of the many known
subunits.
Nischarin bears limited resemblance to identified proteins with a well-known functions. Only two close homologues of Nischarin have been reported in the DNA data bases. The human homologue of Nischarin has been described as a putative imidazoline receptor (
Nischarin is expressed in many cell types and is found both in the adult mouse and in the developing embryo. Higher amounts of Nischarin are found in neuronal-derived cell lines than in epithelial cells or fibroblasts, but some Nischarin is expressed in all of these cell types. Western blotting of various cell lines for endogenous Nischarin revealed proteins of two distinct sizes; in some cells, an 190 kD form is found that comigrates with expressed Nischarin. However, in neuronal cells, a larger form of the protein is seen. The basis for this difference is currently unknown, but may reflect cell-type specific alternative splicing, use of alternate start codons, or posttranslational modification.
Immunofluorescence and biochemical fractionation studies indicate that Nischarin is largely a soluble cytosolic protein. It is clear from fluorescence microscopy studies that Nischarin is not concentrated in vinculin-rich focal contacts. Although, one might expect a protein that interacts with 5ß1 integrin to be localized to focal contacts, this is not always the case. For example, members of the TM4 family of proteins clearly interact specifically with certain integrins, but TM4 proteins are not found in "classic" focal contacts (
5ß1 integrin and Nischarin that might affect its subcellular distribution.
It seems clear that Nischarin can selectively bind to the cytoplasmic domain of the 5 integrin subunit, based both upon two-hybrid analysis and coimmunoprecipitation of full-length Nischarin with native
5ß1 in mammalian cells. However, at any given time, only a small fraction of the total Nischarin in a cell is likely to be bound to the integrin, since most Nischarin is found in the cytosolic fraction. This type of situation is often seen in signaling pathways, where only a minority of a cytosolic effector molecule associates with its membrane-bound partner molecule. The well-known association between Ras and Raf-1 is a good example, where Raf is primarily found in the cytosol, despite its clear ability to interact with membrane-bound Ras (
Overexpression of full-length Nischarin results in major changes in cell behavior and also affects cytoskeletal organization. The most dramatic aspect is the profound inhibition of cell migration caused by Nischarin. At this point, it is unclear whether the reduced cell migration observed in the "wounding" and transwell assays used here is due to a reduction in innate motility or to an impairment of directional movement (-subunit selective. Thus,
5ß1-dependent migration on fibronectin is inhibited far more strongly than migration on other substrata mediated by other integrins.
The overexpression of Nischarin in certain fibroblasts leads to substantial changes in focal contact and actin filament organization. Thus, Nischarin-transfected REF cells display fewer linear stress fibers and a reduction in mature, vinculin-positive focal contacts. Instead, the actin filaments form unusual "basket" structures around the cell periphery. These effects are clearly seen in well spread fibroblasts such as REF and WI-38 cells, but are much less apparent in cell lines such as 3T3 and Cos. The dramatic effects of Nischarin on cell migration and actin filament organization described here may be due, at least in part, to the fact that the transfected molecule is expressed at substantially higher levels than the normal amount of endogenous Nischarin. However, even the somewhat skewed effects triggered by overexpression may provide important clues in eventually ascertaining the physiological role of Nischarin.
The observed effects of Nischarin on cell motility and cytoskeletal organization suggested that Nischarin might impact the pathways used by some Rho-family GTPases to regulate individual pools of actin filaments (
Lately, the mechanistic basis underlying integrin-mediated cell movement has received a good deal of attention. It is clear that FAK is a key regulator of cell migration in most cells (
In summary, we have identified and characterized a novel protein that we have named Nischarin. This protein can bind selectively to the cytoplasmic tail of the integrin 5 subunit. Overexpression of Nischarin has potent effects in terms of retarding cell migration, and it acts preferentially on migration mediated by the
5ß1 integrin. Nischarin overexpression also influences actin filament organization in some cell types. These effects may be mediated through Nischarin's selective action on pathways regulated by the Rac GTPase. Thus, one important aspect of Nischarin's biological role may be to counterbalance the effects of Rac in promoting directed cell movement.
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: FAK, focal adhesion kinase; GFP, green fluorescent protein; ORF, open reading frame; REF, rat embryonic fibroblast; RIE, rat intestinal epithelial.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We would like to thank Mike Fisher for help with tissue culture work, Dr. Peter Reddig for the GFPNischarin construct and experiments with reporter gene assays, and Dr. Andrew Aplin for the 5- and
2-positive 3T3 cell lines. We also thank Drs. U. Naik and T. Griffith for their suggestions concerning yeast two-hybrid techniques, and Dr. K. Burridge for his critical reading of the manuscript. In addition, the authors thank Brenda Asam for outstanding secretarial assistance.
This work was supported by a grant from the National Institutes of Health to R.L. Juliano (CA 74966).
Submitted: 15 September 2000
Revised: 12 October 2000
Accepted: 12 October 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|