Phalloidin staining of filamentous actin in primary keratinocytes <i>in vitro</i> and developing eyelids <i>in vivo</i>.

<p>Primary keratinocytes were isolated from newborn pups and cultured in either low (60 μM; A, B) or high (1.2 mM; C, D) Ca²⁺ to induce differentiation. (A) Abundant stress fibers are apparent in wild type (+/+) keratinocytes. However, the intensity of phalloidin staining is decreased in LIMK2-deficient keratinocytes (B, −/−). The micrographs in A and B were taken at equal exposures. (C) After stimulation with Ca²⁺ for 24 hours, prominent cortical actin and stress fibers are present in +/+ keratinocytes. (D) Although the overall cell morphology in LIMK2-deficient keratinocytes is comparable to wild type cells, the intensity of cortical phalloidin stain is decreased. (E) Confocal microscopy of wholemount E15.5 ocular adnexa stained with phalloidin reveals intense stress fibers in the +/+ epithelium (ep) adjacent to the basal layer (bl). Cortical actin is also evident in the leading periderm cells (p) in the migrating tip. (F) Although cortical actin is obvious in the −/− periderm cells, stress fibers were not readily apparent in the adjacent epithelium. Scale bar  = 50 μm in A–D and 75 μm in E and F.</p

Ocular anatomy prior to eyelid fusion.

<p>H & E-stained paraffin sections of eyelids obtained from E14.5 (A and B) and E15.5 (C and D) embryos. No phenotypic differences are apparent in <i>Limk2</i> knockout (B) mice compared with control (A). The eyelid of both heterozygous (+/−) and homozygous (−/−) is largely comprised of mesenchyme (m) covered by epithelium (ep). The morphology of periderm (p) cells in the eyelid tip is round in contrast to the flat on the surface of the eyelid. Arrows in A and B point to specialized periderm cells that have changed from a squamous to a rounded morphology in preparation for epithelial sheet migration. A phenotype is clearly observed in E15.5 specimens. (C) The epithelium (ep) in heterozygous mice (+/−) has matured into a sheet of cells behind the rounded peridermal cells (p) that lead the migrating front across the cornea. The morphology of epithelial cells in the basal layer (bl) is clearly distinct from the adjacent mesenchymal cells (m) and the superficial epithelial cells. (D) Although rounded peridermal (p) cells are present in the <i>Limk2</i>-deficient eyelids, there is no evidence of epithelial sheet extension across the cornea. Scale bar  = 50 μm.</p

Generation and characterization of <i>Limk2-</i>mutant mice.

<p>(A) Retroviral gene trap vector VICTR48 (EU676804) was used to produce OmniBank clone OST80053, which contains an insertion within intron 12 of the <i>Limk2</i> gene. The <i>Limk2</i> transcript variants 1–3 (v1–3) represent protein isoforms a-c, respectively, according to current NCBI Reference Sequence annotation for accessions NM_010718.3, NM_173053.1, and NM_001034030.1 (intron/exon numbering based on transcript v1). This mutation would be expected to truncate the <i>Limk2</i> gene product within the kinase domain following coding exon 12, disrupting all reported transcript variants of the <i>Limk2</i> gene. Open boxes denote untranslated exons, filled boxed denote coding exons. Exons that code for the LIM domains are shown in orange, those that code for the PDZ domain are shown in blue, and those that code for the kinase domain are shown in red. LTR, viral long terminal repeat; SA, splice acceptor sequence; <i>neo</i>, neomycin phosphotransferase gene; pA, polyadenylation sequence; <i>Pgk</i>, phosphoglycerate kinase-1 promoter; <i>Btk</i>-SD, Bruton's tyrosine kinase splice donor sequence. *The Limk2 v3 transcript is elsewhere referred to as Limk2t <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047168#pone.0047168-Ikebe1" target="_blank">[45]</a> or tLimk2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047168#pone.0047168-Takahashi2" target="_blank">[46]</a>. (B) RT-PCR expression analysis of <i>Limk2</i> transcripts. Endogenous <i>Limk2</i> transcription was detected in the brain, eye and testis of wild type (+/+) and heterozygous (+/−) mice. No endogenous <i>Limk2</i> expression was detected in homozygous (−/−) tissues. Primers 10F and 14R are complementary to <i>Limk2</i> exons 10 and 14/15, respectively, and amplify a product of 489 nucleotides. (C) Immunoblotting analysis with a rat monoclonal antibody that recognizes mouse LIMK2 residues 145–260, which includes the PDZ domain, revealed LIMK2 in several tissues from wild type mice (+/+) such as spleen, brain, lung and retina. LIMK2 was undetectable in these same tissues isolated from homozygous <i>Limk2</i> (−/−) mutant mice. (D) Mouse primary keratinocytes were cultured from neonatal animals and probed with antibodies that recognize phospho-cofilin, total cofilin or GAPDH. Cell cultures were obtained from several litters to quantify the ratio of p-cofilin to cofilin. There was a statistically significant reduction in p-cofilin levels in the knockout compared to wild type. The difference between the means was 66%±7 (<i>n</i> = 4 per genotype; P<0.0001; student's T test).</p

Ocular anatomy following eyelid fusion but prior to birth.

<p>H & E-stained paraffin sections of Bouin's-fixed specimens obtained at E18.5. (A) Eyelids are fused in wild type embryos (+/+) prior to birth. (B) Eyelids are not fused in <i>Limk2</i>-deficient embryos. With the exception of the open eyelids, the ocular histology is comparable to wild type. (C) Higher magnification view of a wild type eyelid opposition shows fusion of the eyelid epithelium (ep) with the initial stages of keratinization (arrowheads) in the superficial epithelium. The basal layer of epithelium is not involved in fusion (arrows). (D) The epithelium (ep) is apparent above the basal layer (arrow) of the epidermis. However, the cornea (c) is exposed prior to birth in the <i>Limk2</i>-deficient mice. Keratinization (arrowhead) is observed on the palpebral epidermis. Scale bar in A and B  = 500 μm and 100 μm in C and D.</p

Ocular anatomy of newborn mice.

<p>(A) Normal appearance of newborn, PD 1.5 mice. Eyelids are fused and completely cover the ocular surface at this age in wild type mice (+/+). (B) Mice lacking <i>Limk2</i> exhibit an eyelid open at birth (EOB) phenotype. Upper and lower eyelids are separated and the ocular surface (arrow) is exposed. Hematoxylin and Eosin (H & E) stained paraffin sections of formalin-fixed specimens obtained at PD 1.5. (C) Wild type mice exhibit a zone of fusion between the upper and lower eyelids (arrow) that involves only the epithelium and not the basal layer of the epidermis. (D) The upper and lower eyelids are not fused in <i>Limk2</i> deficient mice. Numerous cellular infiltrates can be observed in this section. Scale bar in D = 100 μm.</p

Specification and differentiation of the ocular adnexa at E15.5.

<p>Cryosections were stained with various markers of keratinocyte differentiation. For orientation purposes c  =  cornea and r  =  retina in all images. Keratin 10 (K10) is expressed in papebral epidermis (ep) in wild type (A, +/+) and LIMK2-deficient (B, −/−) eyelids. The conjunctiva (conj.) is negative for K10 expression. An antibody recognizing phosphorylated c-Jun labels nuclei in cells (arrows) at the tip of the eyelid in both +/+ (C) and −/− (D) mice. Keratin 6 (K6) is expressed in epithelial cells at the tip of the eyelids in both +/+ (E) and −/− (F) mice, consistent with induction of a migratory phenotype. Scale bar  = 100 μm.</p

Distribution of LIMK2 in E15.5 ocular adnexa.

<p>(A) Low magnification view of cross sections through the ocular tissue (nuclei appear blue with Hematoxylin stain) in a heterozygous mouse (+/−) immuno-labeled with an anti-Limk2 monoclonal rabbit antibody (brown stain). High levels of LIMK2 are detected in both upper (ul) and lower (ll) eyelids. (B) LIMK2 is not detectable in homozygous (−/−) <i>Limk2</i>-deficient mice. (C) Higher magnification of the upper eyelid (ul) shown in A. Periderm cells (p) in both the epithelial sheet (ep) and cornea (c) express LIMK2. The migrating keratinocytes in the ep and the corneal epithelium contain LIMK2, whereas mesenchyme (m) is not labeled. Note the more intense brown LIMK2-specific signal in the epithelium of the eyelid sheet compared to adjacent palpebral epithelium (pe) or basal layer (bl) of the epidermis. (D) LIMK2 immunoreactivity was not observed in the knockout (−/−) tissue. Scale bar in A and B  = 200 μm and 50 μm in C and D.</p

Expression of <i>Limks</i> in ocular tissues during eyelid closure.

<p><i>In situ</i> hybridization in E15.5 specimens with radioactive riboprobes (as = antisense; s  =  sense) specific for either <i>Limk2</i> or <i>Limk1</i>. (A) High levels of <i>Limk2</i> are detected in the epithelium (ep) in both upper and lower eyelids in wild type mice (+/+). The epithelium in the cornea (c) and differentiated cells in the ganglion cell layer (gcl) of the retina express <i>Limk2</i>. The emerging epithelial sheets at the eyelid tips (arrows) exhibit more intense <i>Limk2</i> hybridization signals compared to adjacent epithelium. (B) Although the intensity of hybridization is decreased in the heterozygous (+/−) specimen, the distribution of <i>Limk2</i> is comparable to that in wild type. Higher levels of <i>Limk2</i> are present in the emerging epithelial sheets (arrows). (C) Homozygous mice, mounted and processed on the same slide as the specimens in A and B, show extremely low levels of hybridization. Three adjacent sections of another wild type E15.5 specimen hybridized with either the antisense <i>Limk2</i> (D), antisense <i>Limk1</i> (E) or sense <i>Limk1</i> (F). <i>Limk2</i> is expressed in the epithelium and gcl, whereas <i>Limk1</i> is detectable in the gcl. The intense signal in the retinal pigment epithelium (rpe in C and F) is an artifact of melanin that appears during dark field illumination and is present in all samples shown. Scale bar  = 100 μm.</p

Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

<div><p>Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an <em>in vitro</em> dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.</p> </div

RPTPγ dephosphorylated itself in vitro.

<p>Wild type RPTPγ or RPTPγ C1060S plasmids were transiently transfected into HEK293F and isolated from lysates with anti c-myc/protein G sepharoses. RPTPγ wild type or C1060S on the protein G sepharose was denatured in 8 M urea to linearized protein as substrates for reaction following. The Purified RPTPγ wild type, C1060s sepharose beads was then incubated with recombinant, active purified RPTPγ cyto enzymes in assay buffer with urea at a final concentration of 0.375 M. Lanes 1 and 2 are RPTPγ WT and RPTPγ C1060S on protein G sepharose in a mock reaction without phosphatase in the same buffer, and lanes 1′and 2′ are RPTPγ wild type and RPTPγ C1060S reacted with RPTPγ cytoplasmic region as phosphatase. Samples were subjected to western blot analysis using anti-phosphotyrosine 4G10 mAb. The densitometry of each band was determined to estimate the extent of removal of phosphotyrosine on the protein. The reacted IgG heavy chain bands were used as internal control to ensure equal loading of samples. It is estimated by densitometry that 75% of phosphotyrosine from RPTPC1060S was removed by addition of RPTPγ enzyme.</p