Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1APC/C^{Cdh1} Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the $APC/C^{Cdh1}$ complex, but not by the $APC/C^{Cdc20}$ complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division

Codanin-1, the protein encoded by the gene mutated in congenital dyserythropoietic anemia type I (CDAN1), is cell cycle-regulated

Codanin-1 is a ubiquitous protein of unknown function, encoded by the gene mutated in congenital dyseritropoietic anemia type 1. The findings of this paper show that codanin-1 is active in S-phase of the cell cycle. See related perspective article on page 599

Gas2l3 is localized to the constriction sites.

<p>(A) Schematic representation of the midbody. (B) HeLa cells expressing EGFP-tagged Gas2l3 were fixed, immunolabeled with either anti-Aurora B or anti-Tubulin, and stained with DAPI. The localization of proteins to the midbody was determined using linescans (stembody and constriction sites are labeled by red and white arrows, respectively). (C) HeLa cells were fixed and immunolabeled with anti-hGas2l3 (serum), and with either anti-Aurora B or anti-Tubulin. Linescans for cells 1 and 2 are plotted. (D) HeLa cells were fixed and immunolabeled with anti-hGas2l3 (serum). The immunofluorescent intensity of the endogenous Gas2l3 at six points across one side of the midbody were measured relative to the width of the intercellular microtubule bridge at these locations (see average and standard error values for <i>N</i> = 5 at the plot, and the scheme underneath). Width and intensity values were normalized to the intercellular bridge width and Gas2l3 intensity at the constriction sites. (E) HeLa cells expressing FLAG-tagged human CHMP4b (top panels), Gas2l3-EGFP and FLAG-CHMP4b (mid panels), or Gas2l3-EGFP and Myc-tagged human Spastin (bottom panels), were fixed and immunolabeled with anti-hGas2l3 (serum [top panels]), anti-FLAG (mid panels), or anti-Myc (bottom panels). Linescans are depicted. We used a Zeiss AxioImager and 100X oil lens for imaging, and ImageJ software for image analysis.</p

Gas2l3 is a cell cycle-regulated protein whose degradation is mediated by the APC/C.

<p>(A) Normalized expression levels of the mouse genes GAS2, GAS2L1, GAS2L3, and CCNA2 in naïve, plasmablast, germinal center (GC), and memory-B cells. Raw data were taken from the GEO database GSE4142. (B) Real-time PCR analysis of human GAS2L3 and CCNA2 expression throughout the cell cycle of HeLa S3 (S3) cells, normalized to ACTB RNA levels at time point 0. Cells were double-thymidine blocked, released, and harvested for RNA extraction every 2 hrs for 24 hrs. Cell cycle progression of the synchronous populations (measured by propidium-iodide (PI) staining followed by FACS analysis) is depicted under the plot. (C) HEK293 (293) cells overexpressing human Gas2l3 or Gas2l3-EGFP were harvested for Western blot analysis with custom-made polyclonal hGas2l3 antibodies (serum). A cross-reactive band is noted (asterisk) in the transfected and untransfected (UT) cells. Forty µg extracts made from transfected cells and 120 µg extracts made from untransfected cells were assayed. (D) Left: 293 cells were transfected with the Gas2l3 expression vector. After 24 hrs, cells were treated with MG132 for 5 hrs and subjected to Western blotting with Gas2l3 (serum) and Actin antibodies (loading control). Right: Human Gas2l3 was expressed in rabbit reticulocytes supplemented with radiolabeled (³⁵S) methionine. <i>In vitro</i> transcribed/translated (IVT) Gas2l3 product was incubated in G1 extracts of S3 cells in the presence of MG132 or DMSO (control). Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (E) ³⁵S-labeled hGeminin, hTome-1, and hGas2l3 IVT products were incubated in G1-phase S3 cell extracts supplemented with either buffer, the C-terminus of hEmi1, or hSecurin. Time-dependent degradation was assayed by SDS-PAGE and autoradiography. (F) Left: The putative D-boxes of human Gas2l3 are depicted. Right: Arg at position 1 and Leu at position 4 of each putative D-box were substituted with Gly and Val, respectively. The degradation of the four D-box mutants (DM1–DM4) was assayed as described in (D). (G) 293 cells were cotransfected with Cdh1 (+) or empty vector (-), and with either Gas2l3 or its mutant derivate Gas2l3-DM4, at a 4∶1 ratio, respectively. After 30 hrs cells were harvested for Western blotting with anti-hGas2l3 (serum) and anti-Tubulin (control).</p

Gas2l3 is a Cdk1 target.

<p>(A) The phosphorylation of Securin, Tome-1 (positive controls), and Gas2l3 was assayed in 293 late mitotic extracts [see (B) for details] in the presence of the Cdk1 inhibitors RO-3306 or roscovitine (+), or DMSO (-). Because Securin is naturally degraded in this extract system, Securin phosphorylation was assayed in the presence of MG132. (B) 293 cells were cotransfected with Myc-tagged Gas2l3 and either non-degradable Cyclin B1 or empty vector to generate asynchronous (US) and late mitotic (late M) cell populations overexpressing Gas2l3. The cells were harvested after 24 hrs for Western blotting (with the depicted antibodies) and analysis by liquid chromatography-mass spectrometry (LC-MS/MS). The samples were separated by SDS-PAGE and the area of interest, as determined by Western blotting, was excised for in-gel digestion following established protocols. The resulting peptide mixture was analyzed using a Q Exactive mass spectrometer (Thermo Scientific) equipped with a nanoflow UPLC system (Eksigent). The LC-MS data were searched against a human protein sequence database (Uniprot/Swissprot, canonical and isoform sequence data) using ProteinPilot (v4.5, AB Sciex), allowing for biological modifications/phosphorylation emphasis. Only peptide-spectrum matches above a 1% FDR cut-off were considered for further analysis. The amino acid sequence of two phosphopeptides is depicted.</p

Gas2l3 degradation is mediated by APC/C^Cdh1, but not by APC/C^Cdc20.

<p>(A) Incubation of IVT-generated Tome-1, Geminin, Gas2l3, and Gas2l3-DM4 in <i>X. laevis</i> interphase egg extracts (right) supplemented with recombinant Cdh1 (+) or buffer (-), or in extracts driven into mitosis (left) by preincubation with recombinant non-degradable Cyclin B1 protein [Δ90 (23)]. (B) APC/C^Cdc20-active extracts were made from 293 cells arrested in late mitosis by overexpression of His-tagged non-degradable hCyclin B1. Degradation of IVT-generated positive (Geminin) and negative (Tome-1) controls and of Gas2l3 was assayed in extracts supplemented with mock (-) or dominant negative UbcH10 (Ubch10^DN). Time-dependent degradation was assayed by SDS-PAGE and autoradiography.</p

High levels of Gas2l3 interfere with cell abscission.

<p>(A) HeLa cells were transfected with vectors expressing Gas2l3-EGFP, Gas2l3-DM4-EGFP, or EGFP (control). Thirty-two hrs post-transfection, cells were fixed and immunolabeled, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057532#pone-0057532-g004" target="_blank">Figure 4C</a>. The slides were scanned using a Zeiss AxioImager upright microscope and 100X oil lens for imaging. Transfected (green) cells were counted in order to detect 500 positive daughter cell pairs connected by a midbody (positives). The proportion of attached daughter cells equals 500/<i>N</i>, where <i>N</i> = the number of transfected cells (<i>N</i> - 500 =  the number of negatives). (B) HeLa cells were transfected with vectors expressing EGFP-tagged Gas2l3 (wt) or Gas2l3-DM4. Thirty-two hours post-transfection, cells were fixed, immunolabeled with anti-MKLP1 (stembody marker) and anti-Tubulin, and stained with DAPI. We used a Zeiss AxioImager Z1 upright microscope and 100X oil lens for imaging.</p