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Gas2l3, a novel constriction site-associated protein whose regulation is mediated by the APC/C Cdh1 complex.

Pe'er T, Lahmi R, Sharaby Y, Chorni E, Noach M, Vecsler M, Zlotorynski E, Steen H, Steen JA, Tzur A - PLoS ONE (2013)

Bottom Line: 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.We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

View Article: PubMed Central - PubMed

Affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

ABSTRACT
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.

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High levels of Gas2l3 interfere with cell abscission.(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 Figure 4C. 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/N, where N = the number of transfected cells (N - 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.
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pone-0057532-g005: High levels of Gas2l3 interfere with cell abscission.(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 Figure 4C. 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/N, where N = the number of transfected cells (N - 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.

Mentions: In a population of cells growing exponentially, the proportion of daughter cells still attached by a midbody is expected to be low due to the relatively short duration of cytokinesis and cell division with respect to the total length of the mammalian cell cycle. In fixed HeLa cells, we measured this proportion to be 3.305% in cells transfected with EGFP as control (Figure 5A). This proportion increased over fourfold in cells overexpressing EGFP-tagged Gas2l3. Moreover, this notable phenotype was even more prevalent for the D-box mutant Gas2l3, where nearly 20% of the transfected cells were attached by an intercellular bridge (detected by the midbody marker MKLP1 [10]). This is equivalent to >6 times the frequency found in the EGFP-transfected cells, and is significantly more frequent (by 34%) in comparison to cells expressing the w.t. Gas2l3-EGFP (p-value [Gas2l3 w.t. vs. DM4] = 1.9533×10−5, calculated by the Fisher’s exact test) (Figure 5A). Interestingly, nearly all daughter-cell pairs overexpressing Gas2l3 were in interphase (evident by their decondensed chromatin, their shape, and the presence of nuclei), fully adherent, and although they were separated from each other – some by a large distance of over 50 microns (e.g., cell 6 DM4 in Figure 5B) – they were still attached by a thin, long, and often abnormally structured microtubule bridge. For example, in cell 5 DM4 (Figure 5B), a midbody remnant, marked by the midbody ring protein MKLP1, is absorbed in one of the two daughter cells (see the white arrow and the matching magnified images), yet a thin and aberrant Tubulin bridge connecting these cells is present. These results suggest that high levels of Gas2l3 appear to permit cytokinesis but prevent cell abscission.


Gas2l3, a novel constriction site-associated protein whose regulation is mediated by the APC/C Cdh1 complex.

Pe'er T, Lahmi R, Sharaby Y, Chorni E, Noach M, Vecsler M, Zlotorynski E, Steen H, Steen JA, Tzur A - PLoS ONE (2013)

High levels of Gas2l3 interfere with cell abscission.(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 Figure 4C. 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/N, where N = the number of transfected cells (N - 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585356&req=5

pone-0057532-g005: High levels of Gas2l3 interfere with cell abscission.(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 Figure 4C. 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/N, where N = the number of transfected cells (N - 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.
Mentions: In a population of cells growing exponentially, the proportion of daughter cells still attached by a midbody is expected to be low due to the relatively short duration of cytokinesis and cell division with respect to the total length of the mammalian cell cycle. In fixed HeLa cells, we measured this proportion to be 3.305% in cells transfected with EGFP as control (Figure 5A). This proportion increased over fourfold in cells overexpressing EGFP-tagged Gas2l3. Moreover, this notable phenotype was even more prevalent for the D-box mutant Gas2l3, where nearly 20% of the transfected cells were attached by an intercellular bridge (detected by the midbody marker MKLP1 [10]). This is equivalent to >6 times the frequency found in the EGFP-transfected cells, and is significantly more frequent (by 34%) in comparison to cells expressing the w.t. Gas2l3-EGFP (p-value [Gas2l3 w.t. vs. DM4] = 1.9533×10−5, calculated by the Fisher’s exact test) (Figure 5A). Interestingly, nearly all daughter-cell pairs overexpressing Gas2l3 were in interphase (evident by their decondensed chromatin, their shape, and the presence of nuclei), fully adherent, and although they were separated from each other – some by a large distance of over 50 microns (e.g., cell 6 DM4 in Figure 5B) – they were still attached by a thin, long, and often abnormally structured microtubule bridge. For example, in cell 5 DM4 (Figure 5B), a midbody remnant, marked by the midbody ring protein MKLP1, is absorbed in one of the two daughter cells (see the white arrow and the matching magnified images), yet a thin and aberrant Tubulin bridge connecting these cells is present. These results suggest that high levels of Gas2l3 appear to permit cytokinesis but prevent cell abscission.

Bottom Line: 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.We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.

View Article: PubMed Central - PubMed

Affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus