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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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Alterations in DPgly-GFP dynamics impair its incorporation into junctions. (A) Wounded DPgly-GFP–expressing A431 cells were imaged at 15 s intervals (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Although all three phases of DP border incorporation were observed, they were temporally delayed. In this movie, phase I (arrows, corresponding to green arrows in Video 9) began after 15 min; the typical onset is 30 min to 1 h after contact. Phase II dots appear to be associated with IF bundles (arrowheads, corresponding to yellow arrows in Video 9). Bar, 20 μm. (B) Fluorescence intensity comparison of DP-GFP (black diamonds) and DPgly-GFP (gray squares) cells imaged under the same conditions, at the same time. DPgly-GFP border fluorescence was hindered compared with DP-GFP, and the intensity increased in a linear fashion, similar to that observed for DPNTP-GFP (Fig. 6 C). (C) Instantaneous velocities at each time point were calculated for particles from DPgly-GFP–expressing cells. A DPgly-GFP particle displayed fluctuating velocities, with a greater number of pause times (73% of frames during tracking) compared with a DP-GFP particle from a paired movie (33%; not depicted). (D) Phase III dynamics in DPgly-GFP–expressing A431 cells imaged at 2-min intervals (Video 10). IF bundles are decorated by the phosphorylation point mutant and are therefore visible in these cells. Yellow, red, and green arrows follow the dynamics of three DP-containing cytoplasmic particles as they joined the maturing border. Bar, 5 μm.
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fig10: Alterations in DPgly-GFP dynamics impair its incorporation into junctions. (A) Wounded DPgly-GFP–expressing A431 cells were imaged at 15 s intervals (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Although all three phases of DP border incorporation were observed, they were temporally delayed. In this movie, phase I (arrows, corresponding to green arrows in Video 9) began after 15 min; the typical onset is 30 min to 1 h after contact. Phase II dots appear to be associated with IF bundles (arrowheads, corresponding to yellow arrows in Video 9). Bar, 20 μm. (B) Fluorescence intensity comparison of DP-GFP (black diamonds) and DPgly-GFP (gray squares) cells imaged under the same conditions, at the same time. DPgly-GFP border fluorescence was hindered compared with DP-GFP, and the intensity increased in a linear fashion, similar to that observed for DPNTP-GFP (Fig. 6 C). (C) Instantaneous velocities at each time point were calculated for particles from DPgly-GFP–expressing cells. A DPgly-GFP particle displayed fluctuating velocities, with a greater number of pause times (73% of frames during tracking) compared with a DP-GFP particle from a paired movie (33%; not depicted). (D) Phase III dynamics in DPgly-GFP–expressing A431 cells imaged at 2-min intervals (Video 10). IF bundles are decorated by the phosphorylation point mutant and are therefore visible in these cells. Yellow, red, and green arrows follow the dynamics of three DP-containing cytoplasmic particles as they joined the maturing border. Bar, 5 μm.

Mentions: Analysis of DPgly-GFP by four-dimensional imaging revealed that the average onset of phase I was delayed and fluorescence intensity suppressed, as compared with DP-GFP in cells with continuous staining along IFs (Fig. 10, A and B) and cells with more particulate staining (not depicted). Phase II was either absent or delayed, with phase II particles appearing on DP-coated filaments, which most likely represented keratin bundles (Fig. 10 A and Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Phase III DPgly-GFP particles exhibited more halting movements and greater pause times than DP-GFP particles (Fig. 10, A, C, and D; and Videos 9 and 10), although instantaneous velocities for DPgly-GFP particles still fell within the range of those exhibited by DP-GFP. A representative particle from Fig. 10 A was stationary from one time point to the next 73% of the time (Fig. 10 C), compared with 33% for a DP-GFP particle from a paired movie (not depicted). 85% of DPgly-GFP particles exhibited pause times, whereas only 30% of DP-GFP particles paused. Collectively, these data suggest that either impairing or enhancing DP–IF interactions delays DP's recruitment to desmosomes.


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)

Alterations in DPgly-GFP dynamics impair its incorporation into junctions. (A) Wounded DPgly-GFP–expressing A431 cells were imaged at 15 s intervals (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Although all three phases of DP border incorporation were observed, they were temporally delayed. In this movie, phase I (arrows, corresponding to green arrows in Video 9) began after 15 min; the typical onset is 30 min to 1 h after contact. Phase II dots appear to be associated with IF bundles (arrowheads, corresponding to yellow arrows in Video 9). Bar, 20 μm. (B) Fluorescence intensity comparison of DP-GFP (black diamonds) and DPgly-GFP (gray squares) cells imaged under the same conditions, at the same time. DPgly-GFP border fluorescence was hindered compared with DP-GFP, and the intensity increased in a linear fashion, similar to that observed for DPNTP-GFP (Fig. 6 C). (C) Instantaneous velocities at each time point were calculated for particles from DPgly-GFP–expressing cells. A DPgly-GFP particle displayed fluctuating velocities, with a greater number of pause times (73% of frames during tracking) compared with a DP-GFP particle from a paired movie (33%; not depicted). (D) Phase III dynamics in DPgly-GFP–expressing A431 cells imaged at 2-min intervals (Video 10). IF bundles are decorated by the phosphorylation point mutant and are therefore visible in these cells. Yellow, red, and green arrows follow the dynamics of three DP-containing cytoplasmic particles as they joined the maturing border. Bar, 5 μm.
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fig10: Alterations in DPgly-GFP dynamics impair its incorporation into junctions. (A) Wounded DPgly-GFP–expressing A431 cells were imaged at 15 s intervals (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Although all three phases of DP border incorporation were observed, they were temporally delayed. In this movie, phase I (arrows, corresponding to green arrows in Video 9) began after 15 min; the typical onset is 30 min to 1 h after contact. Phase II dots appear to be associated with IF bundles (arrowheads, corresponding to yellow arrows in Video 9). Bar, 20 μm. (B) Fluorescence intensity comparison of DP-GFP (black diamonds) and DPgly-GFP (gray squares) cells imaged under the same conditions, at the same time. DPgly-GFP border fluorescence was hindered compared with DP-GFP, and the intensity increased in a linear fashion, similar to that observed for DPNTP-GFP (Fig. 6 C). (C) Instantaneous velocities at each time point were calculated for particles from DPgly-GFP–expressing cells. A DPgly-GFP particle displayed fluctuating velocities, with a greater number of pause times (73% of frames during tracking) compared with a DP-GFP particle from a paired movie (33%; not depicted). (D) Phase III dynamics in DPgly-GFP–expressing A431 cells imaged at 2-min intervals (Video 10). IF bundles are decorated by the phosphorylation point mutant and are therefore visible in these cells. Yellow, red, and green arrows follow the dynamics of three DP-containing cytoplasmic particles as they joined the maturing border. Bar, 5 μm.
Mentions: Analysis of DPgly-GFP by four-dimensional imaging revealed that the average onset of phase I was delayed and fluorescence intensity suppressed, as compared with DP-GFP in cells with continuous staining along IFs (Fig. 10, A and B) and cells with more particulate staining (not depicted). Phase II was either absent or delayed, with phase II particles appearing on DP-coated filaments, which most likely represented keratin bundles (Fig. 10 A and Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200510038/DC1). Phase III DPgly-GFP particles exhibited more halting movements and greater pause times than DP-GFP particles (Fig. 10, A, C, and D; and Videos 9 and 10), although instantaneous velocities for DPgly-GFP particles still fell within the range of those exhibited by DP-GFP. A representative particle from Fig. 10 A was stationary from one time point to the next 73% of the time (Fig. 10 C), compared with 33% for a DP-GFP particle from a paired movie (not depicted). 85% of DPgly-GFP particles exhibited pause times, whereas only 30% of DP-GFP particles paused. Collectively, these data suggest that either impairing or enhancing DP–IF interactions delays DP's recruitment to desmosomes.

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