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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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Ultrastructural analysis: DP-GFP associates with nascent junctions, mature desmosomes, and PKP2-containing particles. Conventionally prepared keratinocytes (A, C, and E) and extracted, immunogold-labeled DP-GFP expressing cells (B, D, F, G, and H) were switched from low to normal calcium to induce early cell contacts. (A and C) Conventional EM of contact sites before the appearance of desmosomes. Electron dense, IF-associated cytoplasmic particles were seen in close proximity to microfilaments (MF) near borders (arrowheads) and microfilament-associated adherens junctions (AJ) at contact sites. (B and D) IF and immunogold-labeled DP-GFP at early cell–cell contact sites; white arrows indicate zone of contact. Cytoplasmic DP-GFP particles of varying size were observed near the zone of contact. (E) Conventional EM of a mature desmosome. (F) A mature desmosome with DP-GFP labeling (18-nm gold) at the plaque and along IF bundles labeled for keratin18 (10-nm gold). (G) DP-GFP (10-nm gold) and PKP2 (18-nm gold) at an early border. DP-GFP was observed in association with single or sparse IF at early contact sites (bottom right). (H) DP-GFP (10 nm) and PKP2 (18 nm) colocalized in cytoplasmic particles along IF. Bar, 1 μm.
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fig4: Ultrastructural analysis: DP-GFP associates with nascent junctions, mature desmosomes, and PKP2-containing particles. Conventionally prepared keratinocytes (A, C, and E) and extracted, immunogold-labeled DP-GFP expressing cells (B, D, F, G, and H) were switched from low to normal calcium to induce early cell contacts. (A and C) Conventional EM of contact sites before the appearance of desmosomes. Electron dense, IF-associated cytoplasmic particles were seen in close proximity to microfilaments (MF) near borders (arrowheads) and microfilament-associated adherens junctions (AJ) at contact sites. (B and D) IF and immunogold-labeled DP-GFP at early cell–cell contact sites; white arrows indicate zone of contact. Cytoplasmic DP-GFP particles of varying size were observed near the zone of contact. (E) Conventional EM of a mature desmosome. (F) A mature desmosome with DP-GFP labeling (18-nm gold) at the plaque and along IF bundles labeled for keratin18 (10-nm gold). (G) DP-GFP (10-nm gold) and PKP2 (18-nm gold) at an early border. DP-GFP was observed in association with single or sparse IF at early contact sites (bottom right). (H) DP-GFP (10 nm) and PKP2 (18 nm) colocalized in cytoplasmic particles along IF. Bar, 1 μm.

Mentions: Conventional EM revealed the presence of electron dense particles, similar to those previously reported, associated with IFs and aligned with microfilament bundles (Fig. 4, A and C, arrows). During early stages of assembly, immunogold analysis of whole mount material revealed DP-GFP at cell–cell interfaces in the absence of well-formed plaques and in the cytoplasm close to the zone of contact (Fig. 4, B, D, and G). DP-GFP also localized to desmosomes later in the assembly process (Fig. 4 F). In the cytoplasm, DP-GFP was in clusters of varying sizes and was often associated with IF bundles (Fig. 4, B, D, and F–H). PKP2 also appeared at borders early (Fig. 4 G), later in desmosomes, and in cytoplasmic particles of varying size that colocalized with DP (Fig. 4, G and H). Single or sparsely organized IFs inserted into DP-GFP–positive early contacts (Fig. 4 G, bottom right inset), whereas larger bundles associated with more mature plaques (Fig. 4 F).


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)

Ultrastructural analysis: DP-GFP associates with nascent junctions, mature desmosomes, and PKP2-containing particles. Conventionally prepared keratinocytes (A, C, and E) and extracted, immunogold-labeled DP-GFP expressing cells (B, D, F, G, and H) were switched from low to normal calcium to induce early cell contacts. (A and C) Conventional EM of contact sites before the appearance of desmosomes. Electron dense, IF-associated cytoplasmic particles were seen in close proximity to microfilaments (MF) near borders (arrowheads) and microfilament-associated adherens junctions (AJ) at contact sites. (B and D) IF and immunogold-labeled DP-GFP at early cell–cell contact sites; white arrows indicate zone of contact. Cytoplasmic DP-GFP particles of varying size were observed near the zone of contact. (E) Conventional EM of a mature desmosome. (F) A mature desmosome with DP-GFP labeling (18-nm gold) at the plaque and along IF bundles labeled for keratin18 (10-nm gold). (G) DP-GFP (10-nm gold) and PKP2 (18-nm gold) at an early border. DP-GFP was observed in association with single or sparse IF at early contact sites (bottom right). (H) DP-GFP (10 nm) and PKP2 (18 nm) colocalized in cytoplasmic particles along IF. Bar, 1 μm.
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fig4: Ultrastructural analysis: DP-GFP associates with nascent junctions, mature desmosomes, and PKP2-containing particles. Conventionally prepared keratinocytes (A, C, and E) and extracted, immunogold-labeled DP-GFP expressing cells (B, D, F, G, and H) were switched from low to normal calcium to induce early cell contacts. (A and C) Conventional EM of contact sites before the appearance of desmosomes. Electron dense, IF-associated cytoplasmic particles were seen in close proximity to microfilaments (MF) near borders (arrowheads) and microfilament-associated adherens junctions (AJ) at contact sites. (B and D) IF and immunogold-labeled DP-GFP at early cell–cell contact sites; white arrows indicate zone of contact. Cytoplasmic DP-GFP particles of varying size were observed near the zone of contact. (E) Conventional EM of a mature desmosome. (F) A mature desmosome with DP-GFP labeling (18-nm gold) at the plaque and along IF bundles labeled for keratin18 (10-nm gold). (G) DP-GFP (10-nm gold) and PKP2 (18-nm gold) at an early border. DP-GFP was observed in association with single or sparse IF at early contact sites (bottom right). (H) DP-GFP (10 nm) and PKP2 (18 nm) colocalized in cytoplasmic particles along IF. Bar, 1 μm.
Mentions: Conventional EM revealed the presence of electron dense particles, similar to those previously reported, associated with IFs and aligned with microfilament bundles (Fig. 4, A and C, arrows). During early stages of assembly, immunogold analysis of whole mount material revealed DP-GFP at cell–cell interfaces in the absence of well-formed plaques and in the cytoplasm close to the zone of contact (Fig. 4, B, D, and G). DP-GFP also localized to desmosomes later in the assembly process (Fig. 4 F). In the cytoplasm, DP-GFP was in clusters of varying sizes and was often associated with IF bundles (Fig. 4, B, D, and F–H). PKP2 also appeared at borders early (Fig. 4 G), later in desmosomes, and in cytoplasmic particles of varying size that colocalized with DP (Fig. 4, G and H). Single or sparsely organized IFs inserted into DP-GFP–positive early contacts (Fig. 4 G, bottom right inset), whereas larger bundles associated with more mature plaques (Fig. 4 F).

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.

Show MeSH
Related in: MedlinePlus