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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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PKP2 with DP-GFP colocalize in cytoplasmic particles. (A) DP-GFP–expressing SCC9 cells were fixed at 15 or 60 min after a calcium switch. Confocal imaging demonstrated that DP-GFP colocalized with endogenous PKP2, Pg, Dsc2, and PKP3 at cell borders. DP-GFP cytoplasmic particles near forming borders only infrequently colocalized with Pg, PKP3, or Dsc3, but extensively colocalized with PKP2. Bar, 10 μm. (B) Average relative fluorescence intensity of each protein in a population of DP-GFP dots, expressed as a ratio with border fluorescence (indicator of maximum colocalization). (C) DP particles did not colocalize with an intracellular membrane dye. Bar, 10 μm.
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fig3: PKP2 with DP-GFP colocalize in cytoplasmic particles. (A) DP-GFP–expressing SCC9 cells were fixed at 15 or 60 min after a calcium switch. Confocal imaging demonstrated that DP-GFP colocalized with endogenous PKP2, Pg, Dsc2, and PKP3 at cell borders. DP-GFP cytoplasmic particles near forming borders only infrequently colocalized with Pg, PKP3, or Dsc3, but extensively colocalized with PKP2. Bar, 10 μm. (B) Average relative fluorescence intensity of each protein in a population of DP-GFP dots, expressed as a ratio with border fluorescence (indicator of maximum colocalization). (C) DP particles did not colocalize with an intracellular membrane dye. Bar, 10 μm.

Mentions: DP-GFP, DPgly-GFP, and DPNTP-GFP were expressed at the predicted molecular weights in inducible A431 (Fig. 1 B) and transient SCC9 (Fig. 1 C) transfectants. Full-length GFP-tagged proteins were expressed at only 1/7 to 1/13 of the level of endogenous DP. DPNTP was present at ∼1/4 of the level of endogenous DP. Furthermore, the expression level (Fig. 1, B and C) and localization (see Fig. 3) of other desmosomal proteins were not detectably altered. DP-GFP was present in discrete cytoplasmic dots, similar to those previously reported for endogenous DP (Figs. 2–5), and during junction assembly DP-GFP accumulated at borders with a time course comparable to that reported for endogenous protein, where it colocalized in a typical punctate pattern with other desmosome components (Fig. 2 and Fig. 3; Watt et al., 1984; Jones and Goldman, 1985; Green et al., 1987; Pasdar and Nelson, 1988b).


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)

PKP2 with DP-GFP colocalize in cytoplasmic particles. (A) DP-GFP–expressing SCC9 cells were fixed at 15 or 60 min after a calcium switch. Confocal imaging demonstrated that DP-GFP colocalized with endogenous PKP2, Pg, Dsc2, and PKP3 at cell borders. DP-GFP cytoplasmic particles near forming borders only infrequently colocalized with Pg, PKP3, or Dsc3, but extensively colocalized with PKP2. Bar, 10 μm. (B) Average relative fluorescence intensity of each protein in a population of DP-GFP dots, expressed as a ratio with border fluorescence (indicator of maximum colocalization). (C) DP particles did not colocalize with an intracellular membrane dye. Bar, 10 μm.
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Related In: Results  -  Collection

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fig3: PKP2 with DP-GFP colocalize in cytoplasmic particles. (A) DP-GFP–expressing SCC9 cells were fixed at 15 or 60 min after a calcium switch. Confocal imaging demonstrated that DP-GFP colocalized with endogenous PKP2, Pg, Dsc2, and PKP3 at cell borders. DP-GFP cytoplasmic particles near forming borders only infrequently colocalized with Pg, PKP3, or Dsc3, but extensively colocalized with PKP2. Bar, 10 μm. (B) Average relative fluorescence intensity of each protein in a population of DP-GFP dots, expressed as a ratio with border fluorescence (indicator of maximum colocalization). (C) DP particles did not colocalize with an intracellular membrane dye. Bar, 10 μm.
Mentions: DP-GFP, DPgly-GFP, and DPNTP-GFP were expressed at the predicted molecular weights in inducible A431 (Fig. 1 B) and transient SCC9 (Fig. 1 C) transfectants. Full-length GFP-tagged proteins were expressed at only 1/7 to 1/13 of the level of endogenous DP. DPNTP was present at ∼1/4 of the level of endogenous DP. Furthermore, the expression level (Fig. 1, B and C) and localization (see Fig. 3) of other desmosomal proteins were not detectably altered. DP-GFP was present in discrete cytoplasmic dots, similar to those previously reported for endogenous DP (Figs. 2–5), and during junction assembly DP-GFP accumulated at borders with a time course comparable to that reported for endogenous protein, where it colocalized in a typical punctate pattern with other desmosome components (Fig. 2 and Fig. 3; Watt et al., 1984; Jones and Goldman, 1985; Green et al., 1987; Pasdar and Nelson, 1988b).

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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