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Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

Godsel LM, Hsieh SN, Amargo EV, Bass AE, Pascoe-McGillicuddy LT, Huen AC, Thorne ME, Gaudry CA, Park JK, Myung K, Goldman RD, Chew TL, Green KJ - J. Cell Biol. (2005)

Bottom Line: Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s.DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively.Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

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DPgly-GFP associates strongly with IF and is insensitive to forskolin-induced junction recruitment. (A) SCC9 cells were transiently transfected with both full-length DP-RFP and DPgly-GFP. Although DP-RFP was largely at cell borders, DPgly-GFP more prominently decorated IF. Bar, 20 μm. (B) Cells were fractionated into cytoplasmic (C), Triton X-100 soluble (S), and triton insoluble (I) fractions. DPgly-GFP in the insoluble fraction was 5× greater than DP-GFP. (C) SCC9 cells stably expressing DP-GFP or DPgly-GFP were treated with 100 μM forskolin for 16 h. DP-GFP fluorescence increased at cell borders and was more organized with forskolin treatment. No difference in border fluorescence intensity was observed in cells expressing DPgly-GFP. Average fluorescence intensities for a fixed cell population are depicted graphically to the right of representative fluorescence images (*, P < 0.00001). Error bars are SEM. Bar, 20 μm.
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fig8: DPgly-GFP associates strongly with IF and is insensitive to forskolin-induced junction recruitment. (A) SCC9 cells were transiently transfected with both full-length DP-RFP and DPgly-GFP. Although DP-RFP was largely at cell borders, DPgly-GFP more prominently decorated IF. Bar, 20 μm. (B) Cells were fractionated into cytoplasmic (C), Triton X-100 soluble (S), and triton insoluble (I) fractions. DPgly-GFP in the insoluble fraction was 5× greater than DP-GFP. (C) SCC9 cells stably expressing DP-GFP or DPgly-GFP were treated with 100 μM forskolin for 16 h. DP-GFP fluorescence increased at cell borders and was more organized with forskolin treatment. No difference in border fluorescence intensity was observed in cells expressing DPgly-GFP. Average fluorescence intensities for a fixed cell population are depicted graphically to the right of representative fluorescence images (*, P < 0.00001). Error bars are SEM. Bar, 20 μm.

Mentions: Abrogation of DP phosphorylation at Ser2849 by site-specific mutation was previously shown to enhance interactions between the DP COOH terminus and IFs (Stappenbeck et al., 1994; Meng et al., 1997; Fontao et al., 2003). To examine the impact of this mutation on full-length DP, the distribution of transiently transfected DP-RFP and the phosphorylation-deficient mutant DPgly-GFP were compared (Fig. 8 A). DPgly-GFP coaligned extensively with IFs, with some punctate staining at borders, whereas DP-RFP was more prominent at cell borders. DP-RFP exhibited some coalignment with IFs, likely because of heterodimerization with DPgly-GFP. The ratio of DPgly-GFP in the detergent insoluble versus cytosolic pool was approximately fivefold greater than that of DP-GFP (Fig. 8 B). These data support the idea that mutation of Ser2849 enhances the association of DP with IF.


Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

Godsel LM, Hsieh SN, Amargo EV, Bass AE, Pascoe-McGillicuddy LT, Huen AC, Thorne ME, Gaudry CA, Park JK, Myung K, Goldman RD, Chew TL, Green KJ - J. Cell Biol. (2005)

DPgly-GFP associates strongly with IF and is insensitive to forskolin-induced junction recruitment. (A) SCC9 cells were transiently transfected with both full-length DP-RFP and DPgly-GFP. Although DP-RFP was largely at cell borders, DPgly-GFP more prominently decorated IF. Bar, 20 μm. (B) Cells were fractionated into cytoplasmic (C), Triton X-100 soluble (S), and triton insoluble (I) fractions. DPgly-GFP in the insoluble fraction was 5× greater than DP-GFP. (C) SCC9 cells stably expressing DP-GFP or DPgly-GFP were treated with 100 μM forskolin for 16 h. DP-GFP fluorescence increased at cell borders and was more organized with forskolin treatment. No difference in border fluorescence intensity was observed in cells expressing DPgly-GFP. Average fluorescence intensities for a fixed cell population are depicted graphically to the right of representative fluorescence images (*, P < 0.00001). Error bars are SEM. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171300&req=5

fig8: DPgly-GFP associates strongly with IF and is insensitive to forskolin-induced junction recruitment. (A) SCC9 cells were transiently transfected with both full-length DP-RFP and DPgly-GFP. Although DP-RFP was largely at cell borders, DPgly-GFP more prominently decorated IF. Bar, 20 μm. (B) Cells were fractionated into cytoplasmic (C), Triton X-100 soluble (S), and triton insoluble (I) fractions. DPgly-GFP in the insoluble fraction was 5× greater than DP-GFP. (C) SCC9 cells stably expressing DP-GFP or DPgly-GFP were treated with 100 μM forskolin for 16 h. DP-GFP fluorescence increased at cell borders and was more organized with forskolin treatment. No difference in border fluorescence intensity was observed in cells expressing DPgly-GFP. Average fluorescence intensities for a fixed cell population are depicted graphically to the right of representative fluorescence images (*, P < 0.00001). Error bars are SEM. Bar, 20 μm.
Mentions: Abrogation of DP phosphorylation at Ser2849 by site-specific mutation was previously shown to enhance interactions between the DP COOH terminus and IFs (Stappenbeck et al., 1994; Meng et al., 1997; Fontao et al., 2003). To examine the impact of this mutation on full-length DP, the distribution of transiently transfected DP-RFP and the phosphorylation-deficient mutant DPgly-GFP were compared (Fig. 8 A). DPgly-GFP coaligned extensively with IFs, with some punctate staining at borders, whereas DP-RFP was more prominent at cell borders. DP-RFP exhibited some coalignment with IFs, likely because of heterodimerization with DPgly-GFP. The ratio of DPgly-GFP in the detergent insoluble versus cytosolic pool was approximately fivefold greater than that of DP-GFP (Fig. 8 B). These data support the idea that mutation of Ser2849 enhances the association of DP with IF.

Bottom Line: Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s.DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively.Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

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