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A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Xie H, Laird DW, Chang TH, Hu VW - J. Cell Biol. (1997)

Bottom Line: This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m).Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A.The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The George Washington University, DC 20037, USA.

ABSTRACT
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

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A representative set of gap-FRAP experiments examining the resumption of GJIC after cytokinesis. One of a pair of  sister cells (HX1) was photobleached in each experiment. Cells  were scanned at 0.8-min intervals after bleaching. Pseudocolor  fluorescence images of the cells are shown at three time points:  before bleaching, immediately after bleaching, and at ∼3 min after bleaching. The lower curves in the % prebleach fluorescence  vs. time graphs depict the time-dependent fluorescence changes  in the respective photobleached cells. In coupled cell pairs, a decrease in fluorescence in the unbleached cell is also observed (a  and c, upper curves) as expected, as dye is transferred to the photobleached cell. (A) Gap-FRAP on a pair of sister cells shows  that the siblings resumed GJIC after cytokinesis (n = 10). (B)  Octanol inhibited the gap junction–mediated dye transfer between sister cells after cytokinesis. Octanol (0.1%) was added immediately before gap-FRAP analyses (n = 5). (C) Gap-FRAP on  a pair of sister cells shows that the resumption of gap junctions  between sister cells could not be blocked by cycloheximide treatment. Mitotic cells were reseeded in culture dishes for 3 h in the  presence of 100 μg/ml cycloheximide (n = 5).
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Figure 4: A representative set of gap-FRAP experiments examining the resumption of GJIC after cytokinesis. One of a pair of sister cells (HX1) was photobleached in each experiment. Cells were scanned at 0.8-min intervals after bleaching. Pseudocolor fluorescence images of the cells are shown at three time points: before bleaching, immediately after bleaching, and at ∼3 min after bleaching. The lower curves in the % prebleach fluorescence vs. time graphs depict the time-dependent fluorescence changes in the respective photobleached cells. In coupled cell pairs, a decrease in fluorescence in the unbleached cell is also observed (a and c, upper curves) as expected, as dye is transferred to the photobleached cell. (A) Gap-FRAP on a pair of sister cells shows that the siblings resumed GJIC after cytokinesis (n = 10). (B) Octanol inhibited the gap junction–mediated dye transfer between sister cells after cytokinesis. Octanol (0.1%) was added immediately before gap-FRAP analyses (n = 5). (C) Gap-FRAP on a pair of sister cells shows that the resumption of gap junctions between sister cells could not be blocked by cycloheximide treatment. Mitotic cells were reseeded in culture dishes for 3 h in the presence of 100 μg/ml cycloheximide (n = 5).

Mentions: Cell coupling assays were performed as previously described (Xie and Hu, 1994b) using gap fluorescence redistribution after photobleaching (gapFRAP) techniques developed by Wade et al. (1986). Briefly, cells were stained at room temperature with 7.3 μg/ml carboxyfluorescein diacetate for 15 min. Using an interactive laser cytometer (model ACAS 570; Meridian Instruments, Okemos, MI), selected cells were bleached with 8–14 50-ms pulses of a strong 488-nm laser beam. The bleached cells included rounded-up or dividing mitotic cells remaining in physical contact with nonmitotic cells after vigorously shaking the culture dish (Table I) or one of a pair of sister cells right after cytokinesis (see Fig. 4). Positive controls consisted of bleached nonmitotic cells that were attached to other nonmitotic cells, while negative controls were bleached cells that were not in contact with any other cells. The fluorescence in unbleached cells was also monitored over time and used to correct for background leakage or bleaching of dye during the course of the experiment. The time-dependent recovery of fluorescence after photobleaching was monitored for each cell by repeated laser scannings during the first 3 min after bleaching. Recovery rates represent the percentage of dye recovery/min relative to the initial (prebleach) fluorescence for the respective cells, as determined by the Cell–Cell Communication software provided by Meridian Instruments.


A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Xie H, Laird DW, Chang TH, Hu VW - J. Cell Biol. (1997)

A representative set of gap-FRAP experiments examining the resumption of GJIC after cytokinesis. One of a pair of  sister cells (HX1) was photobleached in each experiment. Cells  were scanned at 0.8-min intervals after bleaching. Pseudocolor  fluorescence images of the cells are shown at three time points:  before bleaching, immediately after bleaching, and at ∼3 min after bleaching. The lower curves in the % prebleach fluorescence  vs. time graphs depict the time-dependent fluorescence changes  in the respective photobleached cells. In coupled cell pairs, a decrease in fluorescence in the unbleached cell is also observed (a  and c, upper curves) as expected, as dye is transferred to the photobleached cell. (A) Gap-FRAP on a pair of sister cells shows  that the siblings resumed GJIC after cytokinesis (n = 10). (B)  Octanol inhibited the gap junction–mediated dye transfer between sister cells after cytokinesis. Octanol (0.1%) was added immediately before gap-FRAP analyses (n = 5). (C) Gap-FRAP on  a pair of sister cells shows that the resumption of gap junctions  between sister cells could not be blocked by cycloheximide treatment. Mitotic cells were reseeded in culture dishes for 3 h in the  presence of 100 μg/ml cycloheximide (n = 5).
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Related In: Results  -  Collection

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Figure 4: A representative set of gap-FRAP experiments examining the resumption of GJIC after cytokinesis. One of a pair of sister cells (HX1) was photobleached in each experiment. Cells were scanned at 0.8-min intervals after bleaching. Pseudocolor fluorescence images of the cells are shown at three time points: before bleaching, immediately after bleaching, and at ∼3 min after bleaching. The lower curves in the % prebleach fluorescence vs. time graphs depict the time-dependent fluorescence changes in the respective photobleached cells. In coupled cell pairs, a decrease in fluorescence in the unbleached cell is also observed (a and c, upper curves) as expected, as dye is transferred to the photobleached cell. (A) Gap-FRAP on a pair of sister cells shows that the siblings resumed GJIC after cytokinesis (n = 10). (B) Octanol inhibited the gap junction–mediated dye transfer between sister cells after cytokinesis. Octanol (0.1%) was added immediately before gap-FRAP analyses (n = 5). (C) Gap-FRAP on a pair of sister cells shows that the resumption of gap junctions between sister cells could not be blocked by cycloheximide treatment. Mitotic cells were reseeded in culture dishes for 3 h in the presence of 100 μg/ml cycloheximide (n = 5).
Mentions: Cell coupling assays were performed as previously described (Xie and Hu, 1994b) using gap fluorescence redistribution after photobleaching (gapFRAP) techniques developed by Wade et al. (1986). Briefly, cells were stained at room temperature with 7.3 μg/ml carboxyfluorescein diacetate for 15 min. Using an interactive laser cytometer (model ACAS 570; Meridian Instruments, Okemos, MI), selected cells were bleached with 8–14 50-ms pulses of a strong 488-nm laser beam. The bleached cells included rounded-up or dividing mitotic cells remaining in physical contact with nonmitotic cells after vigorously shaking the culture dish (Table I) or one of a pair of sister cells right after cytokinesis (see Fig. 4). Positive controls consisted of bleached nonmitotic cells that were attached to other nonmitotic cells, while negative controls were bleached cells that were not in contact with any other cells. The fluorescence in unbleached cells was also monitored over time and used to correct for background leakage or bleaching of dye during the course of the experiment. The time-dependent recovery of fluorescence after photobleaching was monitored for each cell by repeated laser scannings during the first 3 min after bleaching. Recovery rates represent the percentage of dye recovery/min relative to the initial (prebleach) fluorescence for the respective cells, as determined by the Cell–Cell Communication software provided by Meridian Instruments.

Bottom Line: This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m).Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A.The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The George Washington University, DC 20037, USA.

ABSTRACT
Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

Show MeSH
Related in: MedlinePlus