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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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Assembly of DPgly-GFP into desmosomes after a calcium switch is delayed. (A) SCC9 cells transiently expressing DP-GFP or DPgly-GFP (green) were fixed at 10-min intervals after a calcium switch (0–90 min) followed by keratin staining (red). DP-GFP appeared at cell borders within 10 min and became prominent by 40 min. DPgly-GFP appeared weakly at cell borders at 20 min, with a dampened increase in intensity at 40 and 60 min. Bar, 10 μm. (B) Comparison of fluorescence intensities of borders shared by pairs of cells expressing DP-GFP or DPgly-GFP over time. DPgly-GFP exhibited significantly reduced intensities from 10–80 min (P < 0.03), normalizing at 90 min. Error bars are SEM. (C) PKP2 colocalized with DP-GFP in cytoplasmic particles and at cell borders (top) and is found in a punctate pattern along filament-associated DPgly-GFP, and at the tips of filament bundles (bottom). Bar, 5 μm.
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fig9: Assembly of DPgly-GFP into desmosomes after a calcium switch is delayed. (A) SCC9 cells transiently expressing DP-GFP or DPgly-GFP (green) were fixed at 10-min intervals after a calcium switch (0–90 min) followed by keratin staining (red). DP-GFP appeared at cell borders within 10 min and became prominent by 40 min. DPgly-GFP appeared weakly at cell borders at 20 min, with a dampened increase in intensity at 40 and 60 min. Bar, 10 μm. (B) Comparison of fluorescence intensities of borders shared by pairs of cells expressing DP-GFP or DPgly-GFP over time. DPgly-GFP exhibited significantly reduced intensities from 10–80 min (P < 0.03), normalizing at 90 min. Error bars are SEM. (C) PKP2 colocalized with DP-GFP in cytoplasmic particles and at cell borders (top) and is found in a punctate pattern along filament-associated DPgly-GFP, and at the tips of filament bundles (bottom). Bar, 5 μm.

Mentions: We previously demonstrated that activation of PKA using forskolin leads to the phosphorylation of Ser2849, reducing association of the DP COOH terminus with K8/18-rich IF networks (Stappenbeck et al., 1994). We hypothesized that PKA activation might release full-length DP from IFs, thus generating a larger assembly-competent pool. Supporting this idea, fluorescence intensity at borders increased twofold in forskolin-treated DP-GFP, but not DPgly-GFP cells (Fig. 8 C), without altering DP expression (not depicted), consistent with the idea that PKA promotes DP assembly into desmosomes through the phosphorylation of Ser2849. To test whether the phosphorylation-deficient mutant exhibits altered assembly kinetics, we calculated fluorescence intensity along contacting borders for pairs of DP-GFP– and DPgly-GFP–expressing cells after a calcium switch (Fig. 9, A and B). Between 10 and 20 min, DP-GFP began to accumulate at cell–cell contact sites, and by 40–50 min border staining was extensive. DPgly-GFP particle redistribution was delayed, with obvious fluorescence accumulating only after 50–60 min. By 90 min, junction staining normalized, but DPgly-GFP was also retained along cytoplasmic IFs. These results suggest that increased association with the IF cytoskeleton delays DP recruitment to cell–cell contact sites.


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)

Assembly of DPgly-GFP into desmosomes after a calcium switch is delayed. (A) SCC9 cells transiently expressing DP-GFP or DPgly-GFP (green) were fixed at 10-min intervals after a calcium switch (0–90 min) followed by keratin staining (red). DP-GFP appeared at cell borders within 10 min and became prominent by 40 min. DPgly-GFP appeared weakly at cell borders at 20 min, with a dampened increase in intensity at 40 and 60 min. Bar, 10 μm. (B) Comparison of fluorescence intensities of borders shared by pairs of cells expressing DP-GFP or DPgly-GFP over time. DPgly-GFP exhibited significantly reduced intensities from 10–80 min (P < 0.03), normalizing at 90 min. Error bars are SEM. (C) PKP2 colocalized with DP-GFP in cytoplasmic particles and at cell borders (top) and is found in a punctate pattern along filament-associated DPgly-GFP, and at the tips of filament bundles (bottom). Bar, 5 μm.
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Related In: Results  -  Collection

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fig9: Assembly of DPgly-GFP into desmosomes after a calcium switch is delayed. (A) SCC9 cells transiently expressing DP-GFP or DPgly-GFP (green) were fixed at 10-min intervals after a calcium switch (0–90 min) followed by keratin staining (red). DP-GFP appeared at cell borders within 10 min and became prominent by 40 min. DPgly-GFP appeared weakly at cell borders at 20 min, with a dampened increase in intensity at 40 and 60 min. Bar, 10 μm. (B) Comparison of fluorescence intensities of borders shared by pairs of cells expressing DP-GFP or DPgly-GFP over time. DPgly-GFP exhibited significantly reduced intensities from 10–80 min (P < 0.03), normalizing at 90 min. Error bars are SEM. (C) PKP2 colocalized with DP-GFP in cytoplasmic particles and at cell borders (top) and is found in a punctate pattern along filament-associated DPgly-GFP, and at the tips of filament bundles (bottom). Bar, 5 μm.
Mentions: We previously demonstrated that activation of PKA using forskolin leads to the phosphorylation of Ser2849, reducing association of the DP COOH terminus with K8/18-rich IF networks (Stappenbeck et al., 1994). We hypothesized that PKA activation might release full-length DP from IFs, thus generating a larger assembly-competent pool. Supporting this idea, fluorescence intensity at borders increased twofold in forskolin-treated DP-GFP, but not DPgly-GFP cells (Fig. 8 C), without altering DP expression (not depicted), consistent with the idea that PKA promotes DP assembly into desmosomes through the phosphorylation of Ser2849. To test whether the phosphorylation-deficient mutant exhibits altered assembly kinetics, we calculated fluorescence intensity along contacting borders for pairs of DP-GFP– and DPgly-GFP–expressing cells after a calcium switch (Fig. 9, A and B). Between 10 and 20 min, DP-GFP began to accumulate at cell–cell contact sites, and by 40–50 min border staining was extensive. DPgly-GFP particle redistribution was delayed, with obvious fluorescence accumulating only after 50–60 min. By 90 min, junction staining normalized, but DPgly-GFP was also retained along cytoplasmic IFs. These results suggest that increased association with the IF cytoskeleton delays DP recruitment to cell–cell contact sites.

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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