Midbody localization of vinexin recruits rhotekin to facilitate cytokinetic abscission

<p>Vinexin is a SH3 domain-containing adaptor protein that has diverse roles in cell adhesion, signal transduction, gene regulation and stress granule assembly. In this study, we found that vinexin localizes at the midbody during cell division and facilitates cytokinesis. Knockdown of vinexin in HeLa cells delayed the mitotic cell cycle progression and increased the time of cell abscission and the failure to resolve the cytoplasmic bridge. Midbody-localized vinexin is essential for recruiting rhotekin to this structure for cytokinesis because overexpression of a vinexin mutant without a rhotekin-binding motif or knockdown of rhotekin also impaired cytokinetic abscission and increased the number of cells arrested at the midbody stage. Aberrant expression of vinexin and rhotekin in various cancers has been implicated to promote metastasis because of their functions in cell adhesion and signaling. Our findings reveal a novel role of vinexin and rhotekin in cytokinetic abscission and provide another perspective of how both molecules may affect oncogenic transformation via this fundamental cell cycle process.</p

Arsenite-Activated JNK Signaling Enhances CPEB4-Vinexin Interaction to Facilitate Stress Granule Assembly and Cell Survival

<div><p>Stress granules (SGs) are compartmentalized messenger ribonucleoprotein particles (mRNPs) where translationally repressed mRNAs are stored when cells encounter environmental stress. Cytoplasmic polyadenylation element-binding protein (CPEB)4 is a sequence-specific RNA-binding protein and translational regulator. In keeping with the results obtained from the study of other RNA-binding proteins, we found CPEB4 localized in SGs in various arsenite-treated cells. In this study, we identified that Vinexin, a CPEB4-interacting protein, is a novel component of SGs. Vinexin is a SH3-domain-containing adaptor protein and affects cell migration through its association with Vinculin to localize at focal adhesions (FAs). Unexpectedly, Vinexin is translocated from FAs to SGs under arsenite-induced stress. The recruitment of Vinexin to SGs depends on its interaction with CPEB4 and influences SG formation and cell survival. Arsenite-activated c-Jun N-terminal kinase (JNK) signaling enhances the association between CPEB4 and Vinexin, which consequently facilitates SG localization of Vinexin. Taken together, this study uncovers a novel interaction between a translational regulator and an adaptor protein to influence SG assembly and cell survival.</p></div

Long-term exposure to estrogen enhances chemotherapeutic efficacy potentially through epigenetic mechanism in human breast cancer cells

<div><p>Chemotherapy is the most common clinical option for treatment of breast cancer. However, the efficacy of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen responsive and the levels of ovarian estrogen vary among the breast cancer patients primarily between pre- and post-menopausal age. Whether this age-dependent variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is not known. Therefore, the objective of this study was to evaluate the effects of natural estrogen 17 beta-estradiol (E2) on the efficacy of chemotherapeutic drugs in breast cancer cells. Estrogen responsive MCF-7 and T47D breast cancer cells were long-term exposed to 100 pg/ml estrogen, and using these cells the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased sensitivities of doxorubicin and cisplatin in estrogen-exposed MCF-7 and T47D cells as compared to their respective control cells. Gene expression analysis of cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-aza-2-deoxycytidine and Trichostatin A prior to doxorubicin and cisplatin treatments. The 5-aza-2 deoxycytidine pre-treatment significantly decreased the estrogen-induced efficacy of doxorubicin and cisplatin, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In summary, the results of this study revealed that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the chemotherapy strategies for treatment of breast cancer patients depending on the serum estrogen levels that varies among pre- and post-menopausal age of the patients.</p></div

JNK signaling promotes CPEB4-Vinexin interaction in arsenite-treated cells.

<p>The 293T cells expressing EGFP or EGFP-Vinexin β were treated with ± arsenite and then harvested for immunoprecipitation using the GFP antibody. The precipitated substances were immunoblotted with (A) CPEB4 and GFP antibodies or (B) Vinculin and GFP antibodies. (C) The 293T cells expressing myc-CPEB4 (CP4) and flag-Vinexin β (Vxn β) were treated with ± arsenite for the denoted times and then precipitated with the flag IgG and immunoblotted using myc and flag antibodies. (D) (E) Similarly to (C) except the cells were pre-treated with various kinase inhibitors, PD98059 (PD), SB2035800 (SB) or SP600125 (SP) for 30 min, followed by arsenite stimulation and co-IP to monitor CPEB4-Vinexin interaction. (F) The 293T cells expressing EGFP-Vinexin β, myc-CPEB4 along with the flag-tagged wt or dominant negative (DN)-JNK1 were harvested at the indicated time after arsenite treatment for co-IP assay. The pull-down substances by the GFP antibody were used for western blotting. For (E, F), the results from three independent experiments were quantified and displayed as mean ± s.e.m. (G) The number of TIA-1-positive SGs in approximately 1000 cells treated with ± SP600125 were analyzed using the MetaMorph software and expressed as the mean ± s.e.m. One and two asterisks denote *<i>P</i><0.05 and **<i>P</i><0.01 (Student’s <i>t</i>-test).</p

Vinexin promotes SG formation and assembly.

<p>HeLa cells infected with lentiviruses expressing without (siCtrl) or with Vinexin shRNA (siVxn) were puromycin-selected, treated with ± arsenite and then harvested at different time points for (A) western blotting or (B) TIA-1 immunostaining to detect SGs. The representative images of siCtrl and siVxn cells during (30 min) and recovering from (180 min) arsenite-induced stress are shown. (C) The number of TIA-1-positive SGs in siCtrl and siVxn cells were analyzed and expressed as the mean ± s.e.m. Approximately 1000 cells in each group collected from three independent experiments were analyzed using the MetaMorph software. The significant difference between siCtrl and siVxn groups at each time point was analyzed with Student’s <i>t</i>-test. (D) The percent of survived siCtrl and siVxn cells after arsenite treatment at the indicated time was determined by PrestoBlue viability assay and presented as mean ± s.e.m. from three independent experiments. (E) To test wt or mutant Vinexin in rescuing SG-assembly defect in siVxn cells, the transfected cells were analyzed by western blotting to examine the expression of indicated proteins or TIA-1 immunostaining to score the SG numbers in ∼1000 transfected cells of each group collected from three independent experiments. One and two asterisks denote *<i>P</i><0.05 and **<i>P</i><0.01 (Student’s <i>t</i>-test).</p

Redistribution of Vinexin from FAs to SGs in arsenite-stressed cells.

<p>(A) U2OS cells were treated without (control) or with arsenite ± cycloheximide (CHX) for 30 min prior to immunostaining of Vinexin, Vinculin (FA marker) and TIA-1 (SG marker). Arrow heads and arrows indicate FAs and SGs, respectively. One hundred FAs were randomly selected from ten cell images in each group to quantify the fluorescence intensities of Vinexin and Vinculin. Similarly, a hundred SGs from ten arsenite-treated cells were analyzed for the signals of Vinexin and TIA-1. The top scatter plot shows the reduction of Vinexin signal in FAs after the addition of arsenite. (B) Distribution of Vinexin and CPEB4 in arsenite or heat shock (42°C, 20 min)-treated HeLa cells. Arrows denote colocalization of CPEB4 and Vinexin in SGs. (C) Live imaging of EGFP-Vinexin β and RFP-TIA-1 distribution in HeLa cells treated with arsenite (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107961#pone.0107961.s003" target="_blank">Figure S3</a> for the entire cell images). Arrow heads and arrows indicate FAs and SGs, respectively. Scale: 10 µm.</p

Vinexin interacts with CPEB4 and localizes at SGs in CPEB4-overexpressing cells.

<p>(A) Molecular architectures of mouse Vinexin (mVinexin), showing the sorbin homology (SoHo) and three Src-homology (SH3) motifs. The α, β and γ isoforms vary at the N-terminal end. Using the CPEB4 N-terminus as the bait, a yeast two hybrid (Y2H) screen identified two clones corresponding to the a.a. 482–610 and 483–633 of mVinexin α. The full-length mVinexin β was also shown for positive (+) interaction with CPEB4. N/A: not applicable. (B) Co-immunoprecipitation (Co-IP) assay. The 293T cell lysates containing flag-Vinexin β along with myc-tagged CPEB2, 3 or 4 were precipitated with myc IgG and immunoblotted with flag and myc antibodies. IB: immunoblotting, IP: immunoprecipitation. (C) Similarly, the 293T cell lysates containing myc-CPEB4 (myc-CP4) without (vector) or with flag-Vxn α, β or γ, were precipitated with flag IgG and immunoblotted with flag and myc antibodies. (D) Subcellular distribution of flag-Vxn α and β forms in COS-7 cells in the absence or presence of myc-CPEB4 expression. Arrow heads and arrows denote focal adhesion (FA) and stress granule (SG) structures, respectively. TIA-1 immunostained signal detected by the AlexaFluor 647-conjugated secondary antibody was pseudo-colored in magenta and used as a SG marker. The signal intensities of TIA-1, myc-CPEB4, flag-Vxn α and β in one hundred SGs randomly selected from ten transfected cells were pairwisely compared and plotted in (E). Scale: 10 µm.</p