14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport

14-3-3 proteins regulate the cell cycle and prevent apoptosis by controlling the nuclear and cytoplasmic distribution of signaling molecules with which they interact. Although the majority of 14-3-3 molecules are present in the cytoplasm, we show here that in the absence of bound ligands 14-3-3 homes to the nucleus. We demonstrate that phosphorylation of one important 14-3-3 binding molecule, the transcription factor FKHRL1, at the 14-3-3 binding site occurs within the nucleus immediately before FKHRL1 relocalization to the cytoplasm. We show that the leucine-rich region within the COOH-terminal α-helix of 14-3-3, which had been proposed to function as a nuclear export signal (NES), instead functions globally in ligand binding and does not directly mediate nuclear transport. Efficient nuclear export of FKHRL1 requires both intrinsic NES sequences within FKHRL1 and phosphorylation/14-3-3 binding. Finally, we present evidence that phosphorylation/14-3-3 binding may also prevent FKHRL1 nuclear reimport. These results indicate that 14-3-3 can mediate the relocalization of nuclear ligands by several mechanisms that ensure complete sequestration of the bound 14-3-3 complex in the cytoplasm

Lentiviral Mediated Transgenesis by In Vivo Manipulation of Spermatogonial Stem Cells

This report describes a technique for the generation of transgenic mice by in vivo manipulation of spermatogonial stem cells with a high rate of success. Spermatogonial stem cells (SSCs) in pre-pubescent animals were infected in vivo with recombinant lentiviruses expressing EGFP-f and mated with normal females. All male pre-founder mice produced transgenic pups with an overall success rate of over 60%. The transgene was heritable and the pre-founder mice could be used in multiple mating experiments. This technology could be used to perform overexpression/knockdown screens in vivo using bar-coded lentiviruses, thus permitting the design of genetic screens in the mouse. Further, this technology could be adapted to other laboratory animals resulting in the generation of model systems that closely approximate human development and disease

Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.</p> <p>Methods</p> <p>To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.</p> <p>Results</p> <p>Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (<it>P </it>= 0.041), increased lymph node metastasis (<it>P </it>= 0.001), less differentiation (<it>P </it>= 0.005), increased recurrence (<it>P </it>= 0.038) and shorter survival (<it>P </it>= 0.004) of the patients.</p> <p>Conclusion</p> <p>In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.</p

Transgenesis: Embryo modification to sperm mediated gene transfer

459-466The generation of transgenic animals by conventional transgenic protocols is cumbersome and not very efficient. To improve the efficiency of obtaining transgenic animals, different groups have attempted to genetically modify spermatozoa and these technologies are collectively referred to as sperm mediated gene transfer (SMGT). SMGT technologies involve the modification of either spermatozoa or spermatogonial stem cells that give rise to spermatozoa, followed by either in vitro fertilization or mating with a wild type female to generate transgenic progeny. In addition to the generation of transgenic mice, the use of SMGT technologies has resulted in multiple insights into male reproductive biology. SMGT has bypassed most of the problems associated with the traditional methods of transgenesis and has considerably improved the efficiency of obtaining transgenic animals. Various techniques have been developed by which SMGT can be achieved and this review provides an overview of the evolution of SMGT technology and will indicate how these might be used to further our understanding of mammalian growth and development

MMP7 Is Required to Mediate Cell Invasion and Tumor Formation upon Plakophilin3 Loss

<div><p>Plakophilin3 (PKP3) loss results in increased transformation in multiple cell lines in vitro and increased tumor formation in vivo. A microarray analysis performed in the PKP3 knockdown clones, identified an inflammation associated gene signature in cell lines derived from stratified epithelia as opposed to cell lines derived from simple epithelia. However, in contrast to the inflammation associated gene signature, the expression of MMP7 was increased upon PKP3 knockdown in all the cell lines tested. Using vector driven RNA interference, it was demonstrated that MMP7 was required for in-vitro cell migration and invasion and tumor formation in vivo. The increase in MMP7 levels was due to the increase in levels of the Phosphatase of Regenerating Liver3 (PRL3), which is observed upon PKP3 loss. The results suggest that MMP7 over-expression may be one of the mechanisms by which PKP3 loss leads to increased cell invasion and tumor formation.</p></div

Loss of MMP7 leads to a decrease in transformation in cells lacking PKP3.

<p><b>(A)</b> mRNA prepared from HCT116 derived PKP3 knockdown cells transfected with the vector control (shpkp3-2 + vec) or the MMP7 knockdown construct (shpkp3-2 + shMMP7-1 and shpkp3-2 + MMP7-2) was used as a substrate for reverse transcriptase followed by real time PCR reactions using oligonucleotides specific for MMP7. All expression was normalized to the levels of GAPDH. The fold change is graphed on the Y-axis and the clone name is on the X-axis. Note that MMP7 levels are lowered in the double knockdown clones as compared to the vector control. The standard errors are plotted and student’s t test was performed (* indicates a p value <0.01). <b>(B)</b> 75μg of a whole cell extract (WCE) was resolved on 12% SDS PAGE gels followed by Western blotting with antibodies specific to PKP3. Note that PKP3 levels are lower in clones with a PKP3 knockdown. Western blots for β actin served as a loading control (upper panels). 100μg of acetone precipitated cell supernatants were resolved on 12% SDS PAGE gels followed by Western blotting with antibodies specific to MMP7. Note that MMP7 levels are higher in supernatants prepared from the PKP3 knockdown cells as compared to the vector controls and the levels are lower in the double knockdown clones. The same blot was stained with Ponceau stain to demonstrate equal loading of proteins (lower panels). <b>(C)</b> Scratch wound healing assays were performed on the HCT116 derived vector control (vec), PKP3 knockdown clones (shpkp3-1 and shpkp3-2), shpkp3-2 derived vector control clone (shpkp3-2+vec) and shpkp3-2 derived MMP7 knockdown clones (shpkp3-2+shMMP7-1 and shpkp3-2+shMMP7-2) as described. <b>(D)</b> Matrigel invasion assays were performed in Boyden’s chambers for HCT116 derived vector control cells, PKP3 knockdown clones and the double knockdown clones. The number of cells observed in ten random fields of the membrane for each clone was determined as described in Materials and Methods, representative images of for each clone are shown. The mean and standard deviation of three independent experiments are plotted. Note that loss of PKP3 leads to an increase in invasion as compared to the vector control and this phenotype is reversed in the double knockdown clones. <b>(E)</b> Soft agar colony formation assay was performed with the HCT116 derived vector control and pkp3 knockdown clones, the shpkp3-2 derived double knockdown clones and the shpkp3-2 derived vector control clone. The mean and standard deviation of three independent experiments is plotted. <b>(F)</b> 1x10⁶ cells of the shpkp3-2 derived vector control or double knockdown clones were injected sub-cutaneously into nude mice and allowed to develop tumors. The table shows the number of mice injected with the respective clones and the number of mice among them which were able to develop tumors. Wherever indicated the p value was calculated using a student’s t-test.</p

PKP3 loss leads to the generation of an inflammation associated signature in cell lines derived from stratified epithelia.

<p>The Y-axis in all panels reflects the fold change in transcription, which is calculated as described in the Materials and Methods. The X-axis indicates the clone name. mRNA was prepared from the vector controls (vec) or PKP3 knockdown clones (sh-pkp3-1 and shpkp3-2) derived from either HCT116, FBM or HaCaT cells as indicated. <b>(A)</b> Real time PCR assays were performed using oligonucleotides specific to PKP3 and GAPDH. Relative expression of PKP3 in the HCT116, HaCaT and FBM derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) was compared to the respective vector control clones (vec). Expression of GAPDH has been used for normalization. <b>(B)</b> Real time PCRs were performed using oligonucleotides specific for IL6, SAA1, S100A8, S100A9, CCL2, CBS and GAPDH, in HaCaT and FBM derived PKP3 knockdown clones and the respective vector controls. Expression of GAPDH has been used for normalization. <b>(C)</b> Real time PCR was performed using oligonucleotides specific to MMP7 and GAPDH, with cDNA obtained from the vector control and PKP3 knockdown clones derived from the three cell types under study. Expression of GAPDH has been used for normalization. The standard errors are plotted and student’s t test was performed (* indicates a p value <0.01).</p

MMP7 expression decreases upon inhibition of PRL3 activity.

<p><b>(A)</b> The HCT116 derived PKP3 knockdown clones (shpkp3-1 and shpkp3-2) or the vector control (vec) were treated with either the vehicle control (DMSO) or 5 or 10 μM PRL-3 inhibitor-1(PRL-3i) for 24 hours. The mRNA prepared from the treated cells was used as a substrate for reverse transcriptase followed by real time PCR reactions using oligonucleotides specific for MMP7. All expression was normalized to the levels of GAPDH. The fold change is graphed on the Y-axis and the clone name is on the X-axis. The standard errors are plotted and student’s t test was performed. Note that MMP7 levels are lowered upon treatment with PRL-3 inhibitor. <b>(B)</b> The HCT116 derived vector control (vec) and PKP3 knockdown clones (shpkp3-1 and shpkp3-2) were treated with either DMSO or 10 μM PRL3 inhibitor-1(PRL3i) for 24 hours or 48 hours. The cell supernatants were collected and a100μg of acetone precipitated protein was resolved on 12% SDS PAGE gels followed by Western blotting with antibodies to MMP7. The same blot was stained with Ponceau stain to indicate equal loading. <b>(C)</b> The whole cell lysates of HCT116 derived vector control and plakophilin3 knockdown clones treated with DMSO or PRL3i for 24 hours were resolved on 12% poly-acrylamide gel. This was followed by Western blotting with antibodies to PKP3, β actin and PRL3. The molecular weights of these proteins are indicated in brackets.</p

<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.</span>

669-676The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), <span style="mso-bidi-font-weight: bold">using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo. </span