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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-GFP and PKP2 colocalize in the assembly-competent particles that appear after cell–cell contact. Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals, fixed, and processed for immunofluorescence after cell–cell contact. (A) PKP2 localizes in all cytoplasmic particles that appeared and moved toward the forming border. Representative images illustrate the fates of numbered DP-GFP particles. Merge shows extensive DP and PKP2 colocalization (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). (B) Most phase II particles that moved toward the forming border did not contain PKP3 (Video 5). However, many preexisting particles (blue circle) and particles moving in a retrograde fashion (orange and yellow ovals) did colocalize with PKP3. (C) Most phase II particles including those that moved toward the forming border did not contain Pg. (D) Most phase II particles did not contain Dsc2 (Video 6). Dsc2-containing particles were larger, perinuclear, exhibited random movements, and were often present before contact (pink and blue circles). (E) Many phase II DP-GFP containing particles appeared to be associated with IF. Bar, 10 μm.
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fig5: DP-GFP and PKP2 colocalize in the assembly-competent particles that appear after cell–cell contact. Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals, fixed, and processed for immunofluorescence after cell–cell contact. (A) PKP2 localizes in all cytoplasmic particles that appeared and moved toward the forming border. Representative images illustrate the fates of numbered DP-GFP particles. Merge shows extensive DP and PKP2 colocalization (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). (B) Most phase II particles that moved toward the forming border did not contain PKP3 (Video 5). However, many preexisting particles (blue circle) and particles moving in a retrograde fashion (orange and yellow ovals) did colocalize with PKP3. (C) Most phase II particles including those that moved toward the forming border did not contain Pg. (D) Most phase II particles did not contain Dsc2 (Video 6). Dsc2-containing particles were larger, perinuclear, exhibited random movements, and were often present before contact (pink and blue circles). (E) Many phase II DP-GFP containing particles appeared to be associated with IF. Bar, 10 μm.

Mentions: To directly correlate particle composition with behavior during junction assembly, we performed retrospective analysis. All phase II DP-GFP particles that appeared during imaging, including those that began to move toward the developing intercellular borders, contained PKP2 (Fig. 5 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1), but not Dsc2 (Fig. 5 D and Video 6). Although Pg (Fig. 5 C) and PKP3 (Fig. 5 B and Video 5) were seen in some particles, neither were concentrated to the extent of PKP2 nor was translocation dependent on their presence. In fact, many PKP3- or Dsc2-positive particles were present before cell contact (Fig. 5, B [blue circle] and D [pink circle]) and some moved in a retrograde (Fig. 5 B, orange and yellow ovals) or random (Fig. 5 D, blue circle) motion. Many particles formed in close association with the keratin IF cytoskeleton (Fig. 5 E), although it was not possible to conclude that all particles formed on IFs. Collectively, these studies demonstrate that DP-GFP selectively assembles with PKP2 into precursor particles during cell contact–initiated desmosome assembly.


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-GFP and PKP2 colocalize in the assembly-competent particles that appear after cell–cell contact. Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals, fixed, and processed for immunofluorescence after cell–cell contact. (A) PKP2 localizes in all cytoplasmic particles that appeared and moved toward the forming border. Representative images illustrate the fates of numbered DP-GFP particles. Merge shows extensive DP and PKP2 colocalization (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). (B) Most phase II particles that moved toward the forming border did not contain PKP3 (Video 5). However, many preexisting particles (blue circle) and particles moving in a retrograde fashion (orange and yellow ovals) did colocalize with PKP3. (C) Most phase II particles including those that moved toward the forming border did not contain Pg. (D) Most phase II particles did not contain Dsc2 (Video 6). Dsc2-containing particles were larger, perinuclear, exhibited random movements, and were often present before contact (pink and blue circles). (E) Many phase II DP-GFP containing particles appeared to be associated with IF. Bar, 10 μm.
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fig5: DP-GFP and PKP2 colocalize in the assembly-competent particles that appear after cell–cell contact. Wounded DP-GFP expressing A431 monolayers were imaged at 1-min intervals, fixed, and processed for immunofluorescence after cell–cell contact. (A) PKP2 localizes in all cytoplasmic particles that appeared and moved toward the forming border. Representative images illustrate the fates of numbered DP-GFP particles. Merge shows extensive DP and PKP2 colocalization (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). (B) Most phase II particles that moved toward the forming border did not contain PKP3 (Video 5). However, many preexisting particles (blue circle) and particles moving in a retrograde fashion (orange and yellow ovals) did colocalize with PKP3. (C) Most phase II particles including those that moved toward the forming border did not contain Pg. (D) Most phase II particles did not contain Dsc2 (Video 6). Dsc2-containing particles were larger, perinuclear, exhibited random movements, and were often present before contact (pink and blue circles). (E) Many phase II DP-GFP containing particles appeared to be associated with IF. Bar, 10 μm.
Mentions: To directly correlate particle composition with behavior during junction assembly, we performed retrospective analysis. All phase II DP-GFP particles that appeared during imaging, including those that began to move toward the developing intercellular borders, contained PKP2 (Fig. 5 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1), but not Dsc2 (Fig. 5 D and Video 6). Although Pg (Fig. 5 C) and PKP3 (Fig. 5 B and Video 5) were seen in some particles, neither were concentrated to the extent of PKP2 nor was translocation dependent on their presence. In fact, many PKP3- or Dsc2-positive particles were present before cell contact (Fig. 5, B [blue circle] and D [pink circle]) and some moved in a retrograde (Fig. 5 B, orange and yellow ovals) or random (Fig. 5 D, blue circle) motion. Many particles formed in close association with the keratin IF cytoskeleton (Fig. 5 E), although it was not possible to conclude that all particles formed on IFs. Collectively, these studies demonstrate that DP-GFP selectively assembles with PKP2 into precursor particles during cell contact–initiated desmosome assembly.

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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