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(Investigative Ophthalmology and Visual Science. 2004;45:3955-3963.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0721
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Pdlim2, a Novel PDZ–LIM Domain Protein, Interacts with {alpha}-Actinins and Filamin A

Mario Torrado,1,2,3 Vladimir V. Senatorov,1,2 Ritu Trivedi,1,4 Robert N. Fariss,5 and Stanislav I. Tomarev1

1From the Sections of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology and 5Biological Imaging Core, National Eye Institute, National Institutes of Health, Bethesda, Maryland.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To characterize properties of Pdlim2, a novel PDZ and LIM domain-containing protein.

METHODS. cDNA encoding Pdlim2 was identified in a cDNA library of transcripts expressed in the tissues of the rat eye irido-corneal angle. The expression pattern of the Pdlim2 gene was studied by Northern blot analysis and in situ hybridization. Proteins interacting with Pdlim2 were identified by pull-down assay and mass spectrometry. Intracellular localization of Pdlim2 was investigated by confocal microscopy.

RESULTS. Rat Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal portion and the LIM domain in the C-terminal portion of the protein. The Pdlim2 gene was specifically expressed in the corneal epithelial cells, but not in the corneal stroma and endothelium nor in other ocular tissues. Pdlim2 was also expressed in the lung. In rat corneal and lung extracts, {alpha}-actinin-1, {alpha}-actinin-4, filamin A, and myosin heavy polypeptide 9 were co-immunoprecipitated with Pdlim2. Myosin VI was co-immunoprecipitated with Pdlim2 from corneal but not lung extracts. {alpha}-Actinins were the most abundant among immunoprecipitated proteins. Direct interaction of Pdlim2 with {alpha}-actinins and filamin was confirmed using pull-down assays and gel overlay assay with purified proteins. Pdlim2 and {alpha}-actinins were co-localized mainly to stress fibers after transfection into COS-7 cells. In transfected COS-7 cells, complexes of Pdlim2 and {alpha}-actinin-1 were preferentially located along the basal aspect.

CONCLUSIONS. These results suggest that Pdlim2, like other ALP subfamily members, may act as an adapter that directs other proteins to the cytoskeleton.

The actin cytoskeleton of a eukaryotic cell plays a critical role in cell motility, migration, and maintenance of cell shape and polarity. Actin filaments may form different structures which range from parallel bundles to gel networks. Actin bundles are essential for strong contractile activity, while actin gel networks regulate the diffusion of small molecules (including water), provide elasticity, and are necessary for weak contractions. Actin-binding proteins orchestrate actin filament assembly in response to different stimuli. For example, head-to-tail dimers of {alpha}-actinin cross-link actin filaments into bundles and may connect the actin cytoskeleton to the cell membrane.1 Dimers of another actin-binding protein, filamin, are formed through a dimerization domain located at the C termini of the large (240–280 kDa) polypeptide chains. The N-terminal actin-binding domains of the filamin dimers participate in the formation of a highly viscous actin gel by promoting orthogonal branching and cross-linking of actin filaments.2 This cross-linked three-dimensional network of actin filaments is present mainly in the cortex and at the leading edge of cells. Filamins also participate in the linkage of the actin cytoskeleton to the plasma membrane through their association with different membrane proteins.2 3

Recent studies suggest that proteins containing PDZ and LIM domains may bind the actin cytoskeleton and play important roles in normal development and disease.4 PDZ domains are 80- to 90-amino acid domains that were originally identified as conserved sequence elements within the postsynaptic protein PSD-95, the Drosophila tumor suppressor dlg-A, and epithelial tight junction protein ZO-1.5 PDZ domains are multifunctional protein–protein interaction motifs. The majority of proteins containing PDZ domains are associated with plasma membranes. The LIM domain is a double zinc finger domain and is also thought to be involved in protein–protein interactions. Many proteins containing the LIM domain in their C-terminal have functions related to the cytoskeleton and signal transduction pathways.6

There is a family of proteins that contains an N-terminal PDZ domain and one or more C-terminal LIM domains. The actinin-associated LIM protein (ALP) subfamily members, which include CLP-36 (also known as elfin, CLIM, and Pdlim1),7 8 9 ALP,10 11 and RIL,12 each contain a single LIM domain. The Enigma subfamily members, which include ENH, Enigma/LMP-1,13 and Cypher1/ZASP,14 each have three C-terminal LIM domains.

In the course of our efforts to characterize genes expressed in the rat eye,15 we identified a novel gene encoding a PDZ–LIM protein which is orthologous to recently identified but not characterized mouse and human genes, Pdlim2, which is also known as mystique.16 17 In the present work, we demonstrated that the Pdlim2 gene is specifically expressed in the corneal epithelial cells in the adult rat eye as judged by in situ hybridization. Using pull-down assays we showed that a purified Pdlim2 protein interacts with {alpha}-actinins, filamin, and myosins when incubated with corneal or lung cell extracts. We suggest that the Pdlim2 is a novel protein that may act as an adapter involved in the interaction of other proteins with actin cytoskeleton in the cornea and lung.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Identification of Clone Encoding Pdlim2 Protein
Total RNA was isolated from dissected combined tissues of the Wistar rat eye irido-corneal angle (iris, ciliary body, and trabecular meshwork) using a phenol/guanidine thiocyanate method (RNazol B; Tel-Test, Friendswood, TX). Purification of poly(A)+RNA, cDNA synthesis, size-fractionation, and directional 5'-Sal I-Not I-3' cloning into a vector (pSport1; Invitrogen, Carlsbad, CA) were done as a service by BioServe (Laurel, MD). Two cDNA libraries, containing 1.3 x 106 and 1.8 x 106 independent clones, respectively, were constructed as described.18 The average sizes of the inserts in these libraries were 1.7 kb and 1.4 kb, respectively. Two thousand clones from each library were randomly selected and sequenced from the 5'-end with fluorescent dideoxynucleotides. Sequencing was done as a service by National Intramural Sequencing Center, National Institutes of Health, Gaithersburg, MD. cDNA gx01d10 was identified as a novel cDNA encoding PDZ–LIM protein and sequenced (GenBank accession number AY531526).

Northern Blot Analysis and In Situ Hybridization
Northern blot analysis was performed once as described19 using a cDNA insert of gx01d10 clone as a probe. In situ hybridization was done twice. For in situ hybridization, rat eyes were fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH 7.4) at 4°C overnight and processed for paraffin embedding. Serial sections (6 µm) were hybridized with a specific 33P-labeled riboprobe. To prepare this probe, gx01d10 plasmid was used as a template in a PCR reaction with oligonucleotides 5'-ATAACAGCCAGTCTTCCCAGAG-3' and 5'-AGAGTTCCAGGCATTGAGTAGC-3'. A PCR fragment with a length of 700 nt was cloned into the pCRII-TOPO vector (Invitrogen). An antisense probe was prepared after linearization of the recombinant plasmid with BamHI and transcription in vitro from the T7 promoter using kit (MAXIscript; Ambion, Austin, TX). In situ hybridization, washes, and autoradiography were done using an in situ hybridization kit (mRNAlocator, Ambion) according with manufacturer specifications.

Pull-Down Assays and Mass Spectrometry
A full-length rat Pdlim2 coding sequence was cloned into a bacterial expression vector (pCAL-n-FLAG; Stratagene, La Jolla, CA). Recombinant protein fused to the 5-kDa tag, which included the calmodulin-binding peptide and FLAG peptide, was purified from induced cultures of E. coli cells (Rosetta-gami B(DE3), Novagen, San Diego, CA) using calmodulin affinity resin (Stratagene). Corneas from five adult Wistar rats or approximately 100 mg of rat lung were homogenized in 1 mL of a solution containing 50 mM Tris (pH 8.0), 300 mM NaCl, 5 mM EDTA, 0.4% Triton X-100, 0.4% Tween 20, and a protease inhibitor cocktail tablet (Roche, Indianapolis, IN). Extracts were centrifuged at 15,000g for 20 minutes at 4°C. Five µg of purified Pdlim2 or FLAG-Bacterial alkaline phosphatase fusion protein (Sigma, St. Louis, MO) were added to 0.5 mL of supernatants and incubated for 1 hour at 4°C with constant mixing. Then 50 µL of the anti-FLAG agarose beads (Sigma) were added, and incubation continued overnight in the same conditions. Beads were washed five times with the buffer used for binding. Beads were boiled in 20 µL 2x Laemmli sample buffer (Invitrogen) for 5 minutes and centrifuged briefly. Ten µL of the supernatant were loaded on 10% SDS-polyacrylamide gel. Gels were stained with Colloidal Blue (Invitrogen) and the most prominent bands were cut from the gels. Protein identification was performed as a service by ProtTech, Inc. (Norristown, PA) using the nanoflow liquid chromatography and tandem mass spectrometry (LC-MS/MS) technique (see http://www.prottech.com for details).

Full-length rat {alpha}-actinin-1 and {alpha}-actinin-4 cDNAs (gw08h06 and gw13b12, respectively) were identified in the rat eye angle library. Full-length cDNA for {alpha}-actinin-2 was amplified from rat embryo random primed cDNA. These three cDNAs were cloned into the pcDNA3.1/Myc-His(+)B vector (Invitrogen). [35S]-labeled full-length {alpha}-actinins were synthesized using a rabbit reticulocyte lysate (TnT Rabbit Reticulocyte Lysate System; Promega, Madison, WI). Labeled proteins were purified using G-25 spin columns (Amersham Biosciences, Piscataway, NJ). [35S]-labeled actinins were incubated with approximately 5 µg of the purified Pdlim2 protein for 1 hour at 4°C in 0.5 mL of the binding buffer containing 50 mM Tris (pH 8.0), 200 mM NaCl, and 0.4% Triton X-100 with constant mixing. Then 50 µL of the anti-FLAG beads were added to each tube and incubation continued overnight at 4°C. The agarose beads were washed five times in the binding buffer. Proteins were eluted by boiling in the SDS-PAGE loading buffer and loaded onto SDS-PAGE gels. Gels were dried after electrophoresis and radioactive proteins were visualized by autoradiography. Efficiency of GST-pull down was estimated by comparison with a 10% input lane.

Gel Overlay Binding Assay
A gel overlay binding assay was performed as described.20 In brief, approximately 1 µg of Pdlim2 purified as described in the previous section, {alpha}-actinin from rabbit skeletal muscle (Cytoskeleton, Inc., Denver, CO) and actin from rabbit muscle (Sigma) were separated by PAGE, transferred to PVDF membrane, and washed with PBST (0.2% Tween-20 and PBS, pH 7.4). The membrane was blocked in 10% dry milk prepared in PBST overnight at 4°C and then washed three times with PBST. The membranes were incubated overnight at 4°C with 10 µg/mL chicken gizzard filamin (Research Diagnostics, Flanders, NJ) in a binding buffer (100 mM KCl, 50 mM Tris-HCl [pH 7.4], 1 mM EGTA, 2 mM MgCl2, 2 mM ATP, 0.3 mM DTT, and 0.2% Tween-20). Filters were washed three times with a binding buffer and then incubated for 1 hour with primary monoclonal antibodies against human (Chemicon, Temecula, CA) or chicken (Sigma) filamin diluted 1:1000 in a binding buffer. Filters were washed three times with a binding buffer and incubated for 1 hour with peroxidase-conjugated anti-mouse Ig (Amersham Biosciences) diluted 1:2000. Filters were washed three times in a binding buffer and bound filamin was detected using a chemiluminescent detection system (SuperSignal; Pierce, Rockford, IL).

Intracellular Localization of Pdlim2
Pdlim2 was cloned into vectors (pEGFP-N1 and pEGFP-C1; Clontech, Palo Alto, CA). Pdlim2-EGFP and {alpha}-actinin-Myc constructs were transfected into COS-7 cells as described.21 Cells were fixed 48 hours after transfection in 4% paraformaldehyde for 10 minutes and washed several times in 1x phosphate buffered saline (PBS, pH 7.4). For immunofluorescence, cells were incubated with the monoclonal mouse anti-c-Myc antibody (dilution 1:2000; Sigma) or with the monoclonal antibody against human filamin (dilution 1:100; Chemicon), both in PBS with 0.5% Triton X-100 and 10% Western blotting reagent (Roche) for 1 hour at room temperature. After repetitive washing in PBS, the signals were visualized using Cy-3 conjugated anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) in 1:400 dilution in PBS with 0.5% Triton-100 and 0.2 µg/mL DAPI (Molecular Probes, Eugene, OR). Nontransfected cells or cells transfected with pGFP-C1 construct alone (Clontech) were used as controls. F-actin fibers were stained with phalloidin (Alexa Fluor 568; Molecular Probes) in final concentration 5 u/mL for 30 minutes at room temperature. 2D and 3D images were recorded with a confocal laser scanning system equipped with Nomarski optics (Leica TCS SP2; Leica Microsystems, Exton, PA). Samples were scanned in sequential scan mode to reduce bleed-through artifacts. 2D fluorogram analysis of the red and green pixels within the image was performed to evaluate protein co-localization (software version 1227 by Leica). 3D blind deconvolution and 3D visualization were performed using a confocal configuration system (AutoDeblur/AutoVisualize v. 8.0; AutoQuant Imaging, Inc., Watervliet, NY).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Isolation of the Pdlim2 cDNA
cDNA clone gx01d10 encoding a novel PDZ–LIM protein was identified in a cDNA library prepared from rat eye tissues involved in aqueous humor production and outflow (see Materials and Methods). While this work was in progress, the orthologous mouse (NM_145978) and human (NM_021630) genes were identified in the course of the NIH mammalian gene collection program16 and the Helix Research Institute (Japan) NEDO human sequencing project.17 They were named Pdlim2 and mystique. The rat genome has been recently sequenced,22 and rat Pdlim2 mRNA was predicted by automated computational analysis (XM_214229). We will refer to the gx01d10 rat gene as Pdlim2 throughout this article.

The rat Pdlim2 protein is 349 amino acids long and has a calculated molecular weight of 37.6 kDa. Pdlim2 shows 79% and 93% identity with human and mouse proteins, respectively (Fig. 1A) . Pdlim2 protein belongs to the ALP subfamily of proteins containing the PDZ domain in the N-terminal part and LIM domain in the C-terminal part. Pdlim2 shows 39%, 39%, and 37% identity with other family members CLP36, RIL, and ALP proteins, respectively.



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  		FIGURE 1. Structural characterization of the rat Pdlim2 gene. (A) Comparison of the rat Pdlim2 protein with those of mouse and human. The rat Pdlim2 sequence is shown in full. Only differing amino acids are shown for other sequences. Asterisks (*) mark the gaps that were introduced into the sequences to maximize identity. PDZ and LIM domains are shown in green and light brown, respectively. (B) The exon-intron structure of the rat Pdlim2 gene. The diagram shows the rat Pdlim2 gene drawn approximately to scale. The rightward arrow depicts transcription initiation sites. Exons are numbered by Roman numerals below the diagram.

 
Comparison of gx01d10 cDNA with the rat genome and with rat EST databases demonstrated that the rat Pdlim2 gene is approximately 12 kb long and contains 11 exons (Fig. 1B) . The second exon is alternatively spliced and not present in the gx01d10 clone. Exon I is followed by exon III in this clone.

Pdlim2 Expression Pattern
The expression pattern of Pdlim2 in adult rat tissues was first studied by Northern blot analysis. Rat Pdlim2 was strongly expressed in the cornea and at lower levels in the sclera, lung, and combined tissues of the eye irido-corneal angle (Fig. 2) . The most abundant Pdlim2 transcript had a length of approximately 1.8 kb. The nature of the minor component with a length of approximately 3.8 kb is not clear.



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  		FIGURE 2. Northern blot analysis of Pdlim2 expression in rat tissues. Two µg of total RNA was loaded per lane. Loaded RNA was visualized by staining with ethidium bromide (not shown). Full-size [32P]-labeled rat Pdlim2 cDNA was used as a probe in these experiments. Lane 1, cornea; lane 2, combined trabecular meshwork, iris and, ciliary body; lane 3, sclera; lane 4, retina; lane 5, lens; lane 6, skeletal muscles; lane 7, heart; lane 8, brain; lane 9, liver; lane 10, kidney; lane 11, lung; lane 12, spleen.

 
Distribution of Pdlim2 mRNA in the intact adult rat eye was studied by in situ hybridization. Pdlim2 mRNA was specifically expressed in the corneal epithelial cells but not in other corneal layers (Figs. 3A 3C) . The level of expression of the Pdlim2 gene in other eye tissues was very low compared with that in the corneal epithelial cells (Fig. 3A) .



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  		FIGURE 3. Radioactive in situ hybridization of Pdlim2 antisense probes with rat eye sections. The corneal epithelium shows strong, specific labeling with the Pdlim2 antisense probe (A, C). Right column (B, D) shows hematoxylin staining in the bright field. (C) and (D) represent 100-fold higher magnification over (A) and (B) in the boxed area. Pdlim2 expression in the corneal epithelial cells was high enough to be detectable in the bright field (D). The density of silver grains observed in the corneal stroma and endothelium at high magnification was comparable with that for the background and was significantly lower than in the epithelium (C). Labeling of the lens (A) is an artifact resulting from probe trapped in the fractures that result when the hard adult rat lens is sectioned. Northern blot hybridization confirms that there are very low levels of Pdlim2 mRNA in the lens (Fig. 2 , lane 5).

 
In silico analysis of the Pdlim2 gene expression in mice and human demonstrated that cDNAs corresponding to this gene are present in many cDNA libraries with higher representation in placenta, lung, kidney, prostate, mammary gland, and brain glioblastoma. In eye tissues, Pdlim2 cDNAs were present among the sequenced clones in the human retina, lens, and RPE/choroids, as well as in the human fetal eye libraries.

Identification of Proteins Interacting with Pdlim2
Since PDZ and LIM domains are protein-interacting domains and are involved in protein–protein interactions when present in other proteins, we tested the ability of the Pdlim2 protein to interact with other proteins using a pull-down assay. Total cornea and lung extract were used in these experiments because Pdlim2 was expressed in these tissues (see the previous section). In general, cornea and lung extracts gave rather similar patterns of proteins co-immunoprecipitated with Pdlim2 (Fig. 4) . The most abundant proteins that co-immunoprecipitated with Pdlim2 in both extracts migrated as a broad band with an apparent molecular mass around 100 kDa. This band appeared to contain more than one protein. A band with an apparent molecular mass of approximately 250 kDa was more pronounced in the lung immunoprecipitates than in the corneal ones. Corneal immunoprecipitates contained a relatively weak band with an apparent molecular mass of 150 kDa, not present in the lung immunoprecipitates. Five protein bands were cut from the corneal immunoprecipitate and named C-100, C-102, C-150, C-200, and C-250, according to their apparent molecular weight. Four protein bands were cut from the lung immunoprecipitate and named L-100, L-102, L-200, and L-250. These bands were identified using the nano-LC-MS/MS technique. Band C-100, which was the most abundant protein in the corneal sample, was identified as {alpha}-actinin-4, while closely migrating band C-102 was identified as a mixture of {alpha}-actinin-1 and {alpha}-actinin-4. In the lung samples, the L-100 band contained a mixture of {alpha}-actinin-1 and {alpha}-actinin-4, whereas the L-102 band contained mainly {alpha}-actinin-1. C-200 and L-200 bands were identified as nonmuscle myosin heavy chain IIA (Myh9). C-250 and L-250 bands were identified as filamin A. Corneal-specific C-150 was identified as myosin VI.



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  		FIGURE 4. Identification of proteins interacting with Pdlim2 in corneal and lung extracts. Corneal and lung cell extracts were incubated with 5 µg of CAL-N-FLAG-Pdlim2 fusion protein or with FLAG-Bacterial alkaline phosphatase (BAP). Subsequently, Pdlim2 and associated proteins as well as control BAP and associated proteins were immunoprecipitated with anti-FLAG antibodies and subjected to SDS-PAGE with subsequent Colloidal Blue staining. Indicated corneal (C) and lung (L) bands were cut of the gel and identified using the nano-LC-MS/MS technique. Positions of the molecular weight markers are shown on the right side.

 
Two approaches were used to test direct interaction of Pdlim2 with identified proteins. Interaction of Pdlim2 with different {alpha}-actinins was analyzed using purified Pdlim2 and [35S]-labeled {alpha}-actinins synthesized in vitro in the rabbit reticulocyte system (Fig. 5) . {alpha}-Actinin-2 was included in these experiments to check possible interaction of Pdlim2 with muscle-specific {alpha}-actinins not highly expressed in the corneal epithelial cells. Pdlim2 interacted with all three {alpha}-actinins tested. However, its interactions with {alpha}-actinin-1 appeared to be stronger that its interaction with {alpha}-actinin-2 or {alpha}-actinin-4 (Fig. 5 , compare lanes 1 with lanes 2 or 3).



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  		FIGURE 5. Pdlim2 interacts with muscle and nonmuscle {alpha}-actinins. Pdlim2 was tested for its ability to pull-down 35S-labeled {alpha}-actinin-1 (lane 1), {alpha}-actinin-2 (lane 2), and {alpha}-actinin-4 (lane 3). Efficiency of interactions can be assessed by comparing the amount of protein pulled-down in test lanes (lower panel) and in the 10% input lanes (upper panel). No bands were detected in the absence of Pdlim2 protein (lane 4). These experiments were repeated twice.

 
Direct interaction of Pdlim2 with filamin was tested by the blot overlay assay. Two different anti-filamin antibodies were used in these experiments. Control Western experiments demonstrated that these antibodies do not react with Pdlim2 (Fig. 6C) . Filamin directly interacted with Pdlim2 but not with {alpha}-actinin in the blot overlay assay (Fig. 6B) . Interaction with actin was very weak and hardly detectable (Fig. 6B) which may be explained by the fact that filamin interacts with filamentous but not with monomeric actin. Most probably actin did not efficiently form filaments in the conditions used in these experiments.



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  		FIGURE 6. Pdlim2 directly interacts with filamin. Three identical gels were loaded with approximately 1 µg of indicated proteins. Gel (A) was used for total protein detection with Coomassie Blue; gel (C) was used for Western blot analysis with anti-chicken filamin monoclonal antibody. Gel (B) was used for immunoblot detection of bound filamin. Significant binding of filamin to recombinant Pdlim2 was observed. Experiments with anti-human filamin antibody gave similar results (not shown).

 
Cellular Association of Pdlim2 with {alpha}-Actinins and Filamin A
Cellular association of Pdlim2 with {alpha}-actinins and filamin was studied in COS-7 cells. These cells possess endogenous filamin A and {alpha}-actinins23 and are easy to transfect. Plasmids containing full-length Pdlim2 fused to EGFP at the N- or C-termini were prepared and transfected into COS-7 cells. Transfected cells were then stained with Alexa Fluor 568-labeled phalloidin. The results of these experiments demonstrated that Pdlim2 was associated with phalloidin-labeled actin stress fibers in the cytoplasm (Figs. 7A 7B 7C) . These results did not depend on the position of EGFP in the Pdlim2 fusion construct (not shown). Association of Pdlim2 with stress fibers was also observed after transfection into mouse myoblast C2C12 cells (not shown).



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  		FIGURE 7. Co-localization of Pdlim2 with actin stress fibers and filamin. Pdlim2-EGFP plasmid was transfected into COS-7 cells. Forty-eight hours after transfection, cells were treated with Alexa Fluor 568-labeled phalloidin (A, B, C) or stained with anti-filamin antibodies (D, E, F). (A) and (D), GFP fluorescence; (B) and (E), phalloidin and filamin staining, respectively; (C) and (F), merged images. Images of a single plane close to the basal membrane are shown.

 
Intracellular association of Pdlim2 with different {alpha}-actinins was tested after co-transfection of Pdlim2-EGFP and different {alpha}-actinin-Myc plasmids into COS-7 cells. Pdlim2 and {alpha}-actinin-1 or {alpha}-actinin-2 showed very similar intracellular distribution and were associated with actin stress fibers in most transfected cells (Figs. 8A 8B 8C 8D 8E 8F) . Cells transfected with Pdlim2 and {alpha}-actinin-4 demonstrated slightly different staining patterns compared with Pdlim2 and {alpha}-actinin-1 and {alpha}-actinin-2 staining (Figs. 8G 8H 8I) . Short dispersed fibers were observed together with stress fibers in cells transfected with Pdlim2 and {alpha}-actinin-4. In general, Pdlim2 co-localized with {alpha}-actinin-4 although {alpha}-actinin-4 gave a broader staining compared with Pdlim2.



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  		FIGURE 8. Co-localization of Pdlim2 with different {alpha}-actinins. Pdlim2-EGFP and indicated {alpha}-actinin-Myc plasmids were transfected into COS-7 cells. Cells were stained with the mouse monoclonal anti-Myc antibody 48 hours after transfection. The most typical patterns of staining are shown: left column, GFP fluorescence; middle column, myc-staining; right column, merged images. Image of a single plane close to the basal membrane are shown.

 
Three-dimensional analysis of intracellular localization of Pdlim2 and {alpha}-actinin-1 or {alpha}-actinin-2 showed that Pdlim2 was co-localized exclusively with intracellular fibers running close to the basal cell membrane, as shown in Figure 9 . Whereas {alpha}-actinin-1 and {alpha}-actinin-2 were co-localized with Pdlim2 in these fibers, these proteins were also found in other regions inside the cell. Interestingly, the ends of Pdlim2-{alpha}-actinin-1 fibers in two adjacent cells may be found in juxtaposition at the border between two cells (Fig. 9D) . Three-dimensional analysis of cells transfected with Pdlim2 and {alpha}-actinin-4 demonstrated that short "spike-like" fibers observed in these cells were relatively weakly stained with Pdlim2 and strongly stained with {alpha}-actinin-4.



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  		FIGURE 9. Three-dimensional confocal analysis of intracellular localization of Pdlim2 and {alpha}-actinin-1. (A) Two-dimensional view of two adjacent COS-7 cells co-transfected with Pdlim2 (green) and {alpha}-actinin-1 (red). Cell nuclei are counterstained with DAPI (blue). Three boxed areas of interests were chosen for further analysis as shown in (B, C, D). (B) Triple view (x-y, y-z, x-z) of the first area of interest boxed in (A) after 3D blind deconvolution. Blue fluorescence was omitted for clarity. Note that Pdlim2 is located only in intracellular fibers running on the basal cell membrane, whereas {alpha}-actinin-1 is also found in other cell regions. (C) 3D visualization of intracellular fibers containing Pdlim2 and {alpha}-actinin-1, as shown in the second area of interest boxed in (A). (D) 3D visualization of fibers containing Pdlim2 and {alpha}-actinin-1 at the region at the border between two adjacent cells as shown in the third area of interest boxed in (A). Note that there are very close contacts between Pdlim2/{alpha}-actinin-1 fibers located in two adjacent cells. Filled arrowheads mark intracellular fibers in which Pdlim2 was co-localized with {alpha}-actinin-1. Arrows mark fibers that associated with {alpha}-actinin-1 but not with Pdlim2. Empty arrowheads mark the border between two cells.

 
Co-localization of Pdlim2 with filamin was studied after transfection of COS-7 cells with Pdlim2-EGFP plasmid and staining of the cells with anti-filamin antibodies (Figs. 7D 7E 7F) . Filamin demonstrated preferential localization in the vicinity of the cell membrane but was also present in the association with actin fibers in the cytoplasm and in the perinuclear area (Fig. 7E) . There was no co-localization of Pdlim2 with filamin in the vicinity of the cell membrane but two proteins were found to be co-localized with actin fibers (Fig. 7F) .


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In the present work, we identified and characterized the rat Pdlim2 gene, a novel gene which is preferentially expressed in the rat corneal epithelial cells. Human and mouse orthologs of this gene have been recently identified, but not characterized among cDNA clones sequenced in the course of human and mouse gene collection programs.16 17 The encoded Pdlim2 protein belongs to the ALP subfamily of PDZ–LIM proteins.7 8 9 10 11 12 Although Pdlim2, like all known ALP subfamily members, contains N-terminal PDZ domain and C-terminal LIM domain, Pdlim2 does not have a conserved region, named ZASP-like motif (ZM) between PDZ and LIM domains. ZM consists of 26 conserved residues and is present in both ALP and CLP36 proteins.24

The Pdlim2 gene shows a unique expression pattern when compared with other ALP family members. In the rat tissues, expression of the CLP36 gene was highest in heart, lung, and liver,7 while ALP was most abundantly expressed in the skeletal muscle.10 The mouse RIL gene is expressed in many tissues with the highest levels of expression in lung, brain, ovary, and uterus.12 25 ALP family members are also expressed in the eye tissues and they were present among sequenced clones in the rat and human eye cDNA libraries (http://neibank.nei.nih.gov/index.shtml). One cDNA clone encoding RIL protein was present among sequenced clones in the rat eye angle library.15 Clones encoding human RIL protein were present among the sequenced clones in the human iris, optic nerve, and fetal eye libraries,26 while clones encoding CLP36 cDNA clones were present in human iris, lens, retina, RPE/choroid and fetal cDNA libraries.26 27 28 29 Although we do not have Pdlim2-specific antibodies and cannot test the distribution of the Pdlim2 protein in different tissues directly, we believe that the distribution of Pdlim2 mRNA should reflect the distribution of the Pdlim2 protein and this protein should be preferentially present in the corneal epithelial cells and lung.

All PDZ–LIM domain proteins tested so far are able to interact with other proteins. For example, CLP36 interacts with {alpha}-actinin-1, {alpha}-actinin-2, and {alpha}-actinin-4 via its PDZ domain30 31 32 as well as with kinase Clik1 through the C-terminal part containing the LIM domain.33 RIL protein interacts {alpha}-actinin-125 and zyxin-related protein TRIP634 via the PDZ domain and with protein tyrosine phosphatase PTP-BL35 via the LIM domain. Interaction of ALP with {alpha}-actinin-2 involves both PDZ and ZM domains.10 24 Pdlim2 resembles other PDZ-Lim domain proteins in its ability to interact with {alpha}-actinins. {alpha}-Actinins produced the strongest bands among Pdlim2-interacting proteins in both corneal and lung samples (Fig. 4) . Although we did not quantitate the relative proportion of {alpha}-actinin-1 and {alpha}-actinin-4 in the Pdlim2 co-immunoprecipitates, our data indicate that {alpha}-actinin-4 was more abundant than {alpha}-actinin-1 in the corneal co-immunoprecipitates, whereas these two {alpha}-actinins were present in more equal quantities in the lung co-immunoprecipitates (see Fig. 4 ). This observation may reflect relative abundance of different {alpha}-actinins in adult cornea and lung. {alpha}-Actinin-4 might be significantly more abundant in the adult cornea compared with {alpha}-actinin-1 since {alpha}-actinin-4 mRNA TAGs were significantly more abundant than {alpha}-actinin-1 mRNA TAGs (69 vs. 1 per 62,206 sequenced TAGs, respectively) in the adult mouse corneal SAGE library.36 At the same time, {alpha}-actinin-1 might be more abundant than {alpha}-actinin-4 in adult lung tissues as judged by the abundance of corresponding mRNA TAGs in the adult human lung SAGE library (see http://cgap.nci.nih.gov). Immunostaining of cornea from several mammalian species demonstrated that {alpha}-actinins are preferentially located in the corneal epithelial cells.37 38 Our preliminary results confirmed these observations for adult rat cornea (Senatorov, Fariss and Tomarev, unpublished results, 2004).

Pdlim2 is able to interact not only with {alpha}-actinins but also with several other actin-binding proteins, including filamin A. Although the ability to interact with both {alpha}-actinins and filamins has not been reported for other proteins belonging to the ALP subfamily, several other proteins may interact with both {alpha}-actinins and filamins. N-RAP, an actin binding LIM protein, interacts with {alpha}-actinins, filamin-2, and Krp1.20 Calsarcin-1, calsarcin-2, and calsarcin-3, muscle-specific proteins, interact with {alpha}-actinins, {gamma}-filamin, and telethonin.39 40 Calsarsin-3 also interacts with the PDZ-LIM domain protein ZASP/Cypher/Oracle.40 Interaction of Pdlim2 with filamin did not require other proteins as judged by blot-overlay assay (Fig. 6) . The filamin band was more pronounced in the lung Pdlim2 co-immunoprecipitates compared with those from the cornea. This may reflect the relative abundance of filamin A in these tissues. Analysis of the SAGE libraries data indicated that filamin A mRNA TAGs are as abundant as actinin-4 mRNA TAGs in the human lung library. Filamin A mRNA TAGs were present only once among the sequenced TAGs in the adult mouse corneal library and seven times among the same amount of sequenced TAGs in the mouse postnatal day 9 corneal library.36 Immunostaining of the adult and embryonic day 18.5 rat corneas demonstrated that filamin was located mainly in the stroma in adult cornea but was more prominent in epithelial cells than in the stroma in embryonic cornea (Senatorov, Fariss, and Tomarev, unpublished results, 2004).

Another actin-binding protein, nonmuscle myosin heavy chain IIA (Myh9), was present in the Pdlim2 co-immunoprecipitates of both corneal and lung lysates (Fig. 4) . Myh9 is involved in cytokinesis, cell mobility, cell polarity, the maintenance of cell architecture, cell differentiation41 and in the formation of focal adhesion.42 The mouse Myh9 gene is expressed in most adult tissues tested with high levels of expression in liver, spleen, lung, and kidney.43 At present we do not know whether Pdlim2 may interact with Myh9 directly. We cannot exclude the possibility that Myh9 may directly interact with another protein which was co-immunoprecipitated together with Pdlim2.

Another myosin, myosin VI, was present in the corneal but not lung co-immunoprecipitates with Pdlim2. Myosin VI mRNA TAGs were present five times among sequenced TAGs in the mouse cornea SAGE library36 but were absent in the adult human lung SAGE library. Myosin VI is involved in membrane trafficking, recycling, cell movement, and endocytosis.44 It may have unique cellular functions, since it moves, unlike most other myosins, toward the minus end of actin filaments. Myosin VI has been shown to interact with several proteins including two PDZ domain containing proteins, SAP9745 and GLUT1CBP.46 SAP97 is a synapse-associated protein belonging to the SAP90/PSD-95 subfamily of membrane-associated guanylate kinase homologs. GLUT1CBP (also known as GIPC) is glucose transporter C-terminal binding protein. It is interesting to note that GLUT1CBP, like Pdlim2, is able to interact with both myosin VI and {alpha}-actinin-1.46 As in the case of Myh9, we still do not know whether myosin VI directly interacts with Pdlim2.

Pdlim2 and several Pdlim2-interacting proteins ({alpha}-actinins, myosin VI, Myh9)47 were preferentially associated with stress fibers. However, interactions of Pdlim2 with {alpha}-actinins, myosin VI, and Myh9 were not limited to stress fibers, since Pdlim2 efficiently co-immunoprecipitated the above-mentioned proteins from non-ionic, low detergent corneal and lung cell extracts. Similar results have been previously reported for Clp36 and {alpha}-actinins.30

Although the biological functions of Pdlim2 in the cornea and lung are still not known, some assumptions can be made on the basis of information available for other family members. It has been shown that mice deficient in gene Alp (Alp–/–) gradually develop cardiomyopathy.4 Alp may directly enhance the capacity of {alpha}-actinins to cross-link actin filaments, while the loss of Alp may contribute to destabilization of actin anchorage sites in cardiac muscle.4 Ril protein may modulate actin stress fiber turnover and enhance association of {alpha}-actinin with F-actin.25 Clp36 may serve as a adapter, recruiting Clik1 kinase to actin stress fibers.33 The PDZ domain of Clp36 was essential for interaction with {alpha}-actinins, while the LIM domain was critical for interaction with Clik1. Interaction of Clp36 and Clik1 was highly specific, as no interaction was observed between Clik1 and either Alp or Ril.33

Pdlim2 is unique among the ALP subfamily members in its ability to interact directly with at least two types of actin-binding proteins, {alpha}-actinins and filamin. It may serve as an adapter that brings other proteins to the cytoskeleton and may be involved in the control of the architecture and mechanics of the actin network in the cornea and lung.


   Acknowledgements
 
The authors thank Robert Horowits (National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH) for advice with gel overlay, and Zbynek Kozmik (NEI, NIH) for critical reading of the manuscript.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent senior authors. Back

3 Present affiliations: Institute of Health Sciences, University of La Coruna, Campus de Oza, Edificio El Fortin, La Coruna, Spain; and Back

4 Division of Endocrinology, Central Drug Research Institute, Lucknow, India. Back

Supported by the National Eye Institute Intramural Program.

Submitted for publication June 18, 2004; revised August 2, 2004; accepted August 5, 2004.

Disclosure: M. Torrado, None; V.V. Senatorov, None; R. Trivedi, None; R.N. Fariss, None; S.I. Tomarev, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Stanislav I. Tomarev, Section of Molecular Mechanisms of Glaucoma, Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, DHHS, Bldg. 7, Room 103, Bethesda, MD 20892-0704; tomarevs{at}nei.nih.gov.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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