Limits...
α TAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells

View Article: PubMed Central - PubMed

ABSTRACT

α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu.

No MeSH data available.


αTAT1 downregulation-induced mitotic catastrophe. (a) Cell fate until finishing the first round M phase in time-lapse recordings was traced manually and divided into four groups: (1) entered M phase and error at metaphase or cytokinesis was observed, (2) entered M phase and produced two daughter cells, (3) detached before entering M phase, and (4) attached not yet entering M phase. Time of the anaphase onset was used to represent M-phase time point. For each treatment, data from three independent experiments are pooled and the total cell number at the initial time point is presented on the graphs. (b–d) Representative phase contrast or fluorescence time serial images of sh #1-induced metaphase or cytokinesis error in HeLa cells. (c) Chromosomes visualized by stably expressing histone H2B-strawberry. (e) Statistical analysis of αTAT1 downregulation-induced M phase error. Only cells entering M phase are included in e; metaphase error indicates cells did not move on to anaphase, cytokinesis error indicates cells passed metaphase properly but did not produce two daughter cells. For each treatment, data from three independent experiments are pooled and the total M-phase events recorded are presented on the graphs. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4979442&req=5

fig4: αTAT1 downregulation-induced mitotic catastrophe. (a) Cell fate until finishing the first round M phase in time-lapse recordings was traced manually and divided into four groups: (1) entered M phase and error at metaphase or cytokinesis was observed, (2) entered M phase and produced two daughter cells, (3) detached before entering M phase, and (4) attached not yet entering M phase. Time of the anaphase onset was used to represent M-phase time point. For each treatment, data from three independent experiments are pooled and the total cell number at the initial time point is presented on the graphs. (b–d) Representative phase contrast or fluorescence time serial images of sh #1-induced metaphase or cytokinesis error in HeLa cells. (c) Chromosomes visualized by stably expressing histone H2B-strawberry. (e) Statistical analysis of αTAT1 downregulation-induced M phase error. Only cells entering M phase are included in e; metaphase error indicates cells did not move on to anaphase, cytokinesis error indicates cells passed metaphase properly but did not produce two daughter cells. For each treatment, data from three independent experiments are pooled and the total M-phase events recorded are presented on the graphs. Scale bar, 25 μm.

Mentions: To further understand the fate of deficient cells, we monitored cell growth using time-lapse microscopy. During 36 h observation, cells detached at the interphase or stayed arrested without entering M phase were increased after sh #1 and sh #2 treatment (Figure 4a). For those entering M phase, 93.5% of the control shRNA-treated HeLa cells entered anaphase properly, mostly at about 1 h after starting to round up, whereas only 55.1% and 74.0% of sh #1- and sh #2-treated cells, respectively, did so (Figure 4e). Similar tendency was observed in A549 cells; although 97.2% of sh #1-treated cells still entered anaphase, this reduction was significant in comparison with 100.0% of observed control cells (n=539) and control shRNA-treated cells (n=465) did so (Figure 4e). Cells arrested at metaphase usually stayed rounded for several hours before their eventual detachment (Figure 4b). To verify whether chromosome alignment at metaphase was impaired after αTAT1 downregulation, HeLa cells stably expressing histone H2B-strawberry were used. Results revealed that chromosomes could be aligned at the metaphase plate of deficient cells, however, after sustaining several hours without entering anaphase, more and more chromosomes moved outward followed by cell detachment (Figure 4c). As for the sh #1- and sh #2-treated cells moving on to cytokinesis, the furrow ingression stage was not noticeably affected but 3.7% and 5.7% of HeLa and 11.7% and 5.5% of A549 cells still underwent furrow regression that mostly produced multiploid cells (Figures 4d and e). These characteristics were consistent with mitotic catastrophe, although HeLa and A549 cells showed slightly different susceptibility during cell cycle stages.


α TAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells
αTAT1 downregulation-induced mitotic catastrophe. (a) Cell fate until finishing the first round M phase in time-lapse recordings was traced manually and divided into four groups: (1) entered M phase and error at metaphase or cytokinesis was observed, (2) entered M phase and produced two daughter cells, (3) detached before entering M phase, and (4) attached not yet entering M phase. Time of the anaphase onset was used to represent M-phase time point. For each treatment, data from three independent experiments are pooled and the total cell number at the initial time point is presented on the graphs. (b–d) Representative phase contrast or fluorescence time serial images of sh #1-induced metaphase or cytokinesis error in HeLa cells. (c) Chromosomes visualized by stably expressing histone H2B-strawberry. (e) Statistical analysis of αTAT1 downregulation-induced M phase error. Only cells entering M phase are included in e; metaphase error indicates cells did not move on to anaphase, cytokinesis error indicates cells passed metaphase properly but did not produce two daughter cells. For each treatment, data from three independent experiments are pooled and the total M-phase events recorded are presented on the graphs. Scale bar, 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979442&req=5

fig4: αTAT1 downregulation-induced mitotic catastrophe. (a) Cell fate until finishing the first round M phase in time-lapse recordings was traced manually and divided into four groups: (1) entered M phase and error at metaphase or cytokinesis was observed, (2) entered M phase and produced two daughter cells, (3) detached before entering M phase, and (4) attached not yet entering M phase. Time of the anaphase onset was used to represent M-phase time point. For each treatment, data from three independent experiments are pooled and the total cell number at the initial time point is presented on the graphs. (b–d) Representative phase contrast or fluorescence time serial images of sh #1-induced metaphase or cytokinesis error in HeLa cells. (c) Chromosomes visualized by stably expressing histone H2B-strawberry. (e) Statistical analysis of αTAT1 downregulation-induced M phase error. Only cells entering M phase are included in e; metaphase error indicates cells did not move on to anaphase, cytokinesis error indicates cells passed metaphase properly but did not produce two daughter cells. For each treatment, data from three independent experiments are pooled and the total M-phase events recorded are presented on the graphs. Scale bar, 25 μm.
Mentions: To further understand the fate of deficient cells, we monitored cell growth using time-lapse microscopy. During 36 h observation, cells detached at the interphase or stayed arrested without entering M phase were increased after sh #1 and sh #2 treatment (Figure 4a). For those entering M phase, 93.5% of the control shRNA-treated HeLa cells entered anaphase properly, mostly at about 1 h after starting to round up, whereas only 55.1% and 74.0% of sh #1- and sh #2-treated cells, respectively, did so (Figure 4e). Similar tendency was observed in A549 cells; although 97.2% of sh #1-treated cells still entered anaphase, this reduction was significant in comparison with 100.0% of observed control cells (n=539) and control shRNA-treated cells (n=465) did so (Figure 4e). Cells arrested at metaphase usually stayed rounded for several hours before their eventual detachment (Figure 4b). To verify whether chromosome alignment at metaphase was impaired after αTAT1 downregulation, HeLa cells stably expressing histone H2B-strawberry were used. Results revealed that chromosomes could be aligned at the metaphase plate of deficient cells, however, after sustaining several hours without entering anaphase, more and more chromosomes moved outward followed by cell detachment (Figure 4c). As for the sh #1- and sh #2-treated cells moving on to cytokinesis, the furrow ingression stage was not noticeably affected but 3.7% and 5.7% of HeLa and 11.7% and 5.5% of A549 cells still underwent furrow regression that mostly produced multiploid cells (Figures 4d and e). These characteristics were consistent with mitotic catastrophe, although HeLa and A549 cells showed slightly different susceptibility during cell cycle stages.

View Article: PubMed Central - PubMed

ABSTRACT

α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu.

No MeSH data available.