Limits...
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.

Show MeSH
DPNTP-GFP exhibits altered dynamics and delayed junction assembly. (A) DPNTP-GFP does not colocalize with the keratin IF network. DP-GFP or DPNTP-GFP expressing A431 cells (green) were fixed and stained for keratin (red). Although DP-GFP particles aligned with keratin IF, DPNTP-GFP particles were distributed more randomly (see merged and enlarged regions, right). Bar, 10 μm. (B) Wounded DPNTP-GFP expressing A431 monolayers were imaged at 5-min intervals (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). DPNTP-GFP phase I was comparable to DP-GFP with fluorescence appearing within 5–10 min of contact. Yellow, red, and green arrows follow three DPNTP-containing cytoplasmic phase III particles as they join the forming cell–cell border. Phase II was not observed. Bar, 10 μm. (C) Comparison of DPNTP-GFP (pink squares) and DP-GFP (blue diamonds) fluorescence intensity over time, from paired cells imaged under the same conditions. Although DP-GFP exhibited two waves of border intensity separated by a plateau (Fig. 2), the increase in DPNTP-GFP border fluorescence was more linear with dampened intensity. (D) Schematic depiction of DPNTP-GFP particle trajectory (left) and velocity (right) over time. Colors correspond to the yellow, red, and green arrows designating particles in B. Many particles like the one designated by the green arrow exhibited random movements, traveling a large total distance with fluctuating instantaneous velocities before incorporating. Some particles (yellow and red arrows) exhibited dynamics more similar to DP-GFP, moving directly into the borders without pausing. (E) Wounded DPNTP-GFP expressing A431 were imaged at 1-min intervals (Video 8). Phase I fluorescence appeared within 10 min of contact (green arrow). Phase III particles are marked with yellow arrows. DPNTP-GFP particles exhibited faster, more random dynamics than DP-GFP particles. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171300&req=5

fig6: DPNTP-GFP exhibits altered dynamics and delayed junction assembly. (A) DPNTP-GFP does not colocalize with the keratin IF network. DP-GFP or DPNTP-GFP expressing A431 cells (green) were fixed and stained for keratin (red). Although DP-GFP particles aligned with keratin IF, DPNTP-GFP particles were distributed more randomly (see merged and enlarged regions, right). Bar, 10 μm. (B) Wounded DPNTP-GFP expressing A431 monolayers were imaged at 5-min intervals (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). DPNTP-GFP phase I was comparable to DP-GFP with fluorescence appearing within 5–10 min of contact. Yellow, red, and green arrows follow three DPNTP-containing cytoplasmic phase III particles as they join the forming cell–cell border. Phase II was not observed. Bar, 10 μm. (C) Comparison of DPNTP-GFP (pink squares) and DP-GFP (blue diamonds) fluorescence intensity over time, from paired cells imaged under the same conditions. Although DP-GFP exhibited two waves of border intensity separated by a plateau (Fig. 2), the increase in DPNTP-GFP border fluorescence was more linear with dampened intensity. (D) Schematic depiction of DPNTP-GFP particle trajectory (left) and velocity (right) over time. Colors correspond to the yellow, red, and green arrows designating particles in B. Many particles like the one designated by the green arrow exhibited random movements, traveling a large total distance with fluctuating instantaneous velocities before incorporating. Some particles (yellow and red arrows) exhibited dynamics more similar to DP-GFP, moving directly into the borders without pausing. (E) Wounded DPNTP-GFP expressing A431 were imaged at 1-min intervals (Video 8). Phase I fluorescence appeared within 10 min of contact (green arrow). Phase III particles are marked with yellow arrows. DPNTP-GFP particles exhibited faster, more random dynamics than DP-GFP particles. Bar, 20 μm.

Mentions: To test whether loss of the IF-binding domain alters DP dynamics, time-lapse imaging of DPNTP-GFP was performed in cells at the leading edge of a scrape wound. DPNTP-GFP was present in cytoplasmic particles with a wider size range than those assembled from wild-type DP. Furthermore, although ∼70% of DP-GFP particles colocalized with keratin IFs, only ∼30% of DPNTP-GFP particles colocalized with IF (Fig. 6 A).


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)

DPNTP-GFP exhibits altered dynamics and delayed junction assembly. (A) DPNTP-GFP does not colocalize with the keratin IF network. DP-GFP or DPNTP-GFP expressing A431 cells (green) were fixed and stained for keratin (red). Although DP-GFP particles aligned with keratin IF, DPNTP-GFP particles were distributed more randomly (see merged and enlarged regions, right). Bar, 10 μm. (B) Wounded DPNTP-GFP expressing A431 monolayers were imaged at 5-min intervals (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). DPNTP-GFP phase I was comparable to DP-GFP with fluorescence appearing within 5–10 min of contact. Yellow, red, and green arrows follow three DPNTP-containing cytoplasmic phase III particles as they join the forming cell–cell border. Phase II was not observed. Bar, 10 μm. (C) Comparison of DPNTP-GFP (pink squares) and DP-GFP (blue diamonds) fluorescence intensity over time, from paired cells imaged under the same conditions. Although DP-GFP exhibited two waves of border intensity separated by a plateau (Fig. 2), the increase in DPNTP-GFP border fluorescence was more linear with dampened intensity. (D) Schematic depiction of DPNTP-GFP particle trajectory (left) and velocity (right) over time. Colors correspond to the yellow, red, and green arrows designating particles in B. Many particles like the one designated by the green arrow exhibited random movements, traveling a large total distance with fluctuating instantaneous velocities before incorporating. Some particles (yellow and red arrows) exhibited dynamics more similar to DP-GFP, moving directly into the borders without pausing. (E) Wounded DPNTP-GFP expressing A431 were imaged at 1-min intervals (Video 8). Phase I fluorescence appeared within 10 min of contact (green arrow). Phase III particles are marked with yellow arrows. DPNTP-GFP particles exhibited faster, more random dynamics than DP-GFP particles. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171300&req=5

fig6: DPNTP-GFP exhibits altered dynamics and delayed junction assembly. (A) DPNTP-GFP does not colocalize with the keratin IF network. DP-GFP or DPNTP-GFP expressing A431 cells (green) were fixed and stained for keratin (red). Although DP-GFP particles aligned with keratin IF, DPNTP-GFP particles were distributed more randomly (see merged and enlarged regions, right). Bar, 10 μm. (B) Wounded DPNTP-GFP expressing A431 monolayers were imaged at 5-min intervals (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). DPNTP-GFP phase I was comparable to DP-GFP with fluorescence appearing within 5–10 min of contact. Yellow, red, and green arrows follow three DPNTP-containing cytoplasmic phase III particles as they join the forming cell–cell border. Phase II was not observed. Bar, 10 μm. (C) Comparison of DPNTP-GFP (pink squares) and DP-GFP (blue diamonds) fluorescence intensity over time, from paired cells imaged under the same conditions. Although DP-GFP exhibited two waves of border intensity separated by a plateau (Fig. 2), the increase in DPNTP-GFP border fluorescence was more linear with dampened intensity. (D) Schematic depiction of DPNTP-GFP particle trajectory (left) and velocity (right) over time. Colors correspond to the yellow, red, and green arrows designating particles in B. Many particles like the one designated by the green arrow exhibited random movements, traveling a large total distance with fluctuating instantaneous velocities before incorporating. Some particles (yellow and red arrows) exhibited dynamics more similar to DP-GFP, moving directly into the borders without pausing. (E) Wounded DPNTP-GFP expressing A431 were imaged at 1-min intervals (Video 8). Phase I fluorescence appeared within 10 min of contact (green arrow). Phase III particles are marked with yellow arrows. DPNTP-GFP particles exhibited faster, more random dynamics than DP-GFP particles. Bar, 20 μm.
Mentions: To test whether loss of the IF-binding domain alters DP dynamics, time-lapse imaging of DPNTP-GFP was performed in cells at the leading edge of a scrape wound. DPNTP-GFP was present in cytoplasmic particles with a wider size range than those assembled from wild-type DP. Furthermore, although ∼70% of DP-GFP particles colocalized with keratin IFs, only ∼30% of DPNTP-GFP particles colocalized with IF (Fig. 6 A).

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