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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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GFP-tagged DP expression and localization. (A) GFP-tagged proteins: full-length DP (DP-GFP) with a wild-type Ser residue at position 2849, a phosphorylation point mutant (DPgly-GFP) with a Ser→Gly replacement at position 2849, and a mutant lacking the central rod and IF-binding domain (DPNTP-GFP). (B) A431 cells were treated with DOX to induce DP-GFP, DPgly-GFP, and DPNTP-GFP, which were expressed at 1/7, 1/13, or 1/4 the level of endogenous DP, respectively. GFP-tagged protein expression did not affect Dsg2, Pg, and PKP2. (C) GFP-tagged and endogenous DP from transiently expressing SCC9 cells were separated on 5 or 7.5% gels. Full-length GFP-tagged proteins (αGFP) were expressed at ≤1/5 the level of endogenous DP. Steady-state levels of endogenous DP, Dsg2, Pg, or PKP2 were not affected compared with parental SCC9s (−). Loading control: αvinculin.
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fig1: GFP-tagged DP expression and localization. (A) GFP-tagged proteins: full-length DP (DP-GFP) with a wild-type Ser residue at position 2849, a phosphorylation point mutant (DPgly-GFP) with a Ser→Gly replacement at position 2849, and a mutant lacking the central rod and IF-binding domain (DPNTP-GFP). (B) A431 cells were treated with DOX to induce DP-GFP, DPgly-GFP, and DPNTP-GFP, which were expressed at 1/7, 1/13, or 1/4 the level of endogenous DP, respectively. GFP-tagged protein expression did not affect Dsg2, Pg, and PKP2. (C) GFP-tagged and endogenous DP from transiently expressing SCC9 cells were separated on 5 or 7.5% gels. Full-length GFP-tagged proteins (αGFP) were expressed at ≤1/5 the level of endogenous DP. Steady-state levels of endogenous DP, Dsg2, Pg, or PKP2 were not affected compared with parental SCC9s (−). Loading control: αvinculin.

Mentions: To establish the temporal sequence of DP dynamics and fate during desmosome assembly we generated three COOH-terminally tagged DP-GFP constructs: wild-type DP (DP-GFP), a phosphorylation-deficient DP with a Ser→Gly substitution at residue 2849 (DPgly-GFP), and a truncated DP encompassing the NH2-terminal 584 amino acids (DPNTP-GFP; Fig. 1 A). DPNTP retains the Pg- and PKP-binding domain necessary for incorporation into desmosomes, but lacks the central rod and the COOH-terminal IF-binding domain. The Ser→Gly mutation enhances interactions between the DP COOH terminus and IFs (Stappenbeck et al., 1994; Fontao et al., 2003).


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)

GFP-tagged DP expression and localization. (A) GFP-tagged proteins: full-length DP (DP-GFP) with a wild-type Ser residue at position 2849, a phosphorylation point mutant (DPgly-GFP) with a Ser→Gly replacement at position 2849, and a mutant lacking the central rod and IF-binding domain (DPNTP-GFP). (B) A431 cells were treated with DOX to induce DP-GFP, DPgly-GFP, and DPNTP-GFP, which were expressed at 1/7, 1/13, or 1/4 the level of endogenous DP, respectively. GFP-tagged protein expression did not affect Dsg2, Pg, and PKP2. (C) GFP-tagged and endogenous DP from transiently expressing SCC9 cells were separated on 5 or 7.5% gels. Full-length GFP-tagged proteins (αGFP) were expressed at ≤1/5 the level of endogenous DP. Steady-state levels of endogenous DP, Dsg2, Pg, or PKP2 were not affected compared with parental SCC9s (−). Loading control: αvinculin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171300&req=5

fig1: GFP-tagged DP expression and localization. (A) GFP-tagged proteins: full-length DP (DP-GFP) with a wild-type Ser residue at position 2849, a phosphorylation point mutant (DPgly-GFP) with a Ser→Gly replacement at position 2849, and a mutant lacking the central rod and IF-binding domain (DPNTP-GFP). (B) A431 cells were treated with DOX to induce DP-GFP, DPgly-GFP, and DPNTP-GFP, which were expressed at 1/7, 1/13, or 1/4 the level of endogenous DP, respectively. GFP-tagged protein expression did not affect Dsg2, Pg, and PKP2. (C) GFP-tagged and endogenous DP from transiently expressing SCC9 cells were separated on 5 or 7.5% gels. Full-length GFP-tagged proteins (αGFP) were expressed at ≤1/5 the level of endogenous DP. Steady-state levels of endogenous DP, Dsg2, Pg, or PKP2 were not affected compared with parental SCC9s (−). Loading control: αvinculin.
Mentions: To establish the temporal sequence of DP dynamics and fate during desmosome assembly we generated three COOH-terminally tagged DP-GFP constructs: wild-type DP (DP-GFP), a phosphorylation-deficient DP with a Ser→Gly substitution at residue 2849 (DPgly-GFP), and a truncated DP encompassing the NH2-terminal 584 amino acids (DPNTP-GFP; Fig. 1 A). DPNTP retains the Pg- and PKP-binding domain necessary for incorporation into desmosomes, but lacks the central rod and the COOH-terminal IF-binding domain. The Ser→Gly mutation enhances interactions between the DP COOH terminus and IFs (Stappenbeck et al., 1994; Fontao et al., 2003).

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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