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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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DP accumulation at newly contacting cell–cell borders occurs in three phases. (A) Wounded DP-GFP–expressing A431 monolayers were imaged at 1.5-min intervals. (a) Three phases of DP-GFP dynamics were observed after cell–cell contact. In phase I, fluorescence began to accumulate at sites of contact within ∼3–10 min. In phase II, cytoplasmic particles began to appear at 30–45 min of contact. In phase III, cytoplasmic particles moved toward the maturing border. To the right of the schematic are images of the microscopic field at 0 and 60 min after contact. Colored boxes indicate enlarged regions (b–d; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Bar, 20 μm. (b) Phase I: DP appeared within 7 min of cell–cell contact. (c) Phase II: DP dots appeared de novo in the cytoplasm within 25 min of contact. (d) Phase III: Cytoplasmic DP dots moved in an anterograde direction and incorporated into the forming border (Video 2). (B) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals (Video 3). The green arrow highlights DP that accumulated within 9 min of cell contact (phase I). Cytoplasmic dots appeared upon initiation of cell contact (49 and 64 min; phase II) and translocated to borders (yellow arrows; phase III). Bar, 10 μm. Fluorescence intensity over time was calculated for representative borders from the videos in A and B (C) and from 32 other movies (D). In the graphs two waves of border fluorescence corresponded with the onset of phases I (green arrows) and III (yellow arrows). Phase II particle formation occurred at the beginning of or during the plateau between phases I and III (red arrows). Results are representative of data obtained from >50 movies.
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fig2: DP accumulation at newly contacting cell–cell borders occurs in three phases. (A) Wounded DP-GFP–expressing A431 monolayers were imaged at 1.5-min intervals. (a) Three phases of DP-GFP dynamics were observed after cell–cell contact. In phase I, fluorescence began to accumulate at sites of contact within ∼3–10 min. In phase II, cytoplasmic particles began to appear at 30–45 min of contact. In phase III, cytoplasmic particles moved toward the maturing border. To the right of the schematic are images of the microscopic field at 0 and 60 min after contact. Colored boxes indicate enlarged regions (b–d; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Bar, 20 μm. (b) Phase I: DP appeared within 7 min of cell–cell contact. (c) Phase II: DP dots appeared de novo in the cytoplasm within 25 min of contact. (d) Phase III: Cytoplasmic DP dots moved in an anterograde direction and incorporated into the forming border (Video 2). (B) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals (Video 3). The green arrow highlights DP that accumulated within 9 min of cell contact (phase I). Cytoplasmic dots appeared upon initiation of cell contact (49 and 64 min; phase II) and translocated to borders (yellow arrows; phase III). Bar, 10 μm. Fluorescence intensity over time was calculated for representative borders from the videos in A and B (C) and from 32 other movies (D). In the graphs two waves of border fluorescence corresponded with the onset of phases I (green arrows) and III (yellow arrows). Phase II particle formation occurred at the beginning of or during the plateau between phases I and III (red arrows). Results are representative of data obtained from >50 movies.

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)

DP accumulation at newly contacting cell–cell borders occurs in three phases. (A) Wounded DP-GFP–expressing A431 monolayers were imaged at 1.5-min intervals. (a) Three phases of DP-GFP dynamics were observed after cell–cell contact. In phase I, fluorescence began to accumulate at sites of contact within ∼3–10 min. In phase II, cytoplasmic particles began to appear at 30–45 min of contact. In phase III, cytoplasmic particles moved toward the maturing border. To the right of the schematic are images of the microscopic field at 0 and 60 min after contact. Colored boxes indicate enlarged regions (b–d; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Bar, 20 μm. (b) Phase I: DP appeared within 7 min of cell–cell contact. (c) Phase II: DP dots appeared de novo in the cytoplasm within 25 min of contact. (d) Phase III: Cytoplasmic DP dots moved in an anterograde direction and incorporated into the forming border (Video 2). (B) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals (Video 3). The green arrow highlights DP that accumulated within 9 min of cell contact (phase I). Cytoplasmic dots appeared upon initiation of cell contact (49 and 64 min; phase II) and translocated to borders (yellow arrows; phase III). Bar, 10 μm. Fluorescence intensity over time was calculated for representative borders from the videos in A and B (C) and from 32 other movies (D). In the graphs two waves of border fluorescence corresponded with the onset of phases I (green arrows) and III (yellow arrows). Phase II particle formation occurred at the beginning of or during the plateau between phases I and III (red arrows). Results are representative of data obtained from >50 movies.
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fig2: DP accumulation at newly contacting cell–cell borders occurs in three phases. (A) Wounded DP-GFP–expressing A431 monolayers were imaged at 1.5-min intervals. (a) Three phases of DP-GFP dynamics were observed after cell–cell contact. In phase I, fluorescence began to accumulate at sites of contact within ∼3–10 min. In phase II, cytoplasmic particles began to appear at 30–45 min of contact. In phase III, cytoplasmic particles moved toward the maturing border. To the right of the schematic are images of the microscopic field at 0 and 60 min after contact. Colored boxes indicate enlarged regions (b–d; Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Bar, 20 μm. (b) Phase I: DP appeared within 7 min of cell–cell contact. (c) Phase II: DP dots appeared de novo in the cytoplasm within 25 min of contact. (d) Phase III: Cytoplasmic DP dots moved in an anterograde direction and incorporated into the forming border (Video 2). (B) Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals (Video 3). The green arrow highlights DP that accumulated within 9 min of cell contact (phase I). Cytoplasmic dots appeared upon initiation of cell contact (49 and 64 min; phase II) and translocated to borders (yellow arrows; phase III). Bar, 10 μm. Fluorescence intensity over time was calculated for representative borders from the videos in A and B (C) and from 32 other movies (D). In the graphs two waves of border fluorescence corresponded with the onset of phases I (green arrows) and III (yellow arrows). Phase II particle formation occurred at the beginning of or during the plateau between phases I and III (red arrows). Results are representative of data obtained from >50 movies.
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.

Show MeSH