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The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.

Crespo CL, Vernieri C, Keller PJ, Garrè M, Bender JR, Wittbrodt J, Pardi R - J. Cell. Sci. (2014)

Bottom Line: A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization.However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo.Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy.

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The PAR complex promotes wound-directed migration of myeloid cells in vivo. (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I-SceI integration sites (Sc), in the presence of I-SceI meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry+; PAR) and the endogenous control (Cherry−; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (two-tailed unpaired Student's t-test). See also supplementary material Fig. S1; Movies 1, 2.
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f01: The PAR complex promotes wound-directed migration of myeloid cells in vivo. (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I-SceI integration sites (Sc), in the presence of I-SceI meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry+; PAR) and the endogenous control (Cherry−; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (two-tailed unpaired Student's t-test). See also supplementary material Fig. S1; Movies 1, 2.

Mentions: To determine how PAR-3, PAR-6 and aPKC regulate the directed migration of leukocytes in vivo in a 3D environment, we developed a model of wound-induced inflammatory cell migration in medaka fish, based on live imaging of tissue-resident myeloid cells expressing membrane-tethered YFP [memYFP, using the transgenic line TG(FmpoP::memYFP) as, in medaka, myeloperoxidase (MPO) is expressed in mixed myeloid lineages that also contain sudanophilic material; supplementary material Fig. S1A] (Aghaallaei et al., 2010; Grabher et al., 2007). Because silencing of the PAR components affects the morphogenesis of several embryonic tissues in zebrafish (Horne-Badovinac et al., 2001; Munson et al., 2008; Wei et al., 2004) and the use of morpholinos is not effective in juvenile medaka (∼9–11 days post-fertilization), we adopted a meganuclease-driven, transient transgenesis approach based on the injection of embryos at the one-cell stage (Rembold et al., 2006). We devised a strategy whereby a set of PAR-complex-interfering mutants, expressed under the myeloid-cell-specific Fmpo promoter (FmpoP), were co-injected with a nuclear-localized fluorescent marker (mCherry fused to histone H2A – H2AmCherry), to track the PAR-mutant-expressing cells (Fig. 1A–C) (Souren et al., 2009). Using this established gene expression system, we first determined that the two transgenes were coexpressed in >72% of cells (supplementary material Fig. S1B–D). As the transgenes were expressed in a mosaic fashion, we could directly compare the control subpopulation (H2AmCherry−) with the PAR-mutant-expressing subset (H2AmCherry+), to assess migration-associated parameters during the wound-response within the same animal (Fig. 1A).


The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.

Crespo CL, Vernieri C, Keller PJ, Garrè M, Bender JR, Wittbrodt J, Pardi R - J. Cell. Sci. (2014)

The PAR complex promotes wound-directed migration of myeloid cells in vivo. (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I-SceI integration sites (Sc), in the presence of I-SceI meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry+; PAR) and the endogenous control (Cherry−; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (two-tailed unpaired Student's t-test). See also supplementary material Fig. S1; Movies 1, 2.
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f01: The PAR complex promotes wound-directed migration of myeloid cells in vivo. (A) TG(FmpoP::memYFP) embryos were injected at the one-cell stage with a 1∶1 mixture of DNA coding for H2AmCherry as a nuclear reporter and one of the PAR transgenes, driven by the myeloid-specific Fmpo promoter and flanked by I-SceI integration sites (Sc), in the presence of I-SceI meganuclease. Wounded larvae with mosaic expression of H2AmCherry in tailfin myeloid cells were imaged. The dashed line represents the wound. The inset shows a transgenic cell (Cherry+; PAR) and the endogenous control (Cherry−; CTR). Scale bars: 50 µm (left panel); 10 µm (inset). (B) Domains of interaction between members of the mammalian PAR complex. Connecting lines indicate regions of the proteins that interact with one another. PB1, phagocyte oxidase/Bem1 domain; Zn, Zinc finger motif; Kinase, catalytic domain; CRIB, Cdc42/Rac interactive binding motif; PDZ, PSD-95/Dlg/Zona occludens-1 domain; CR1, conserved region 1; aPKCBR, aPKC-binding region. A predicted coiled-coil region is also shown. (C) Schematics of the constructs used to perturb the function of the PAR complex in myeloid cells. Numbers refer to amino acid positions. *K to W mutation at codon 281. NT, N-terminal domain. (D) 2D tracks of individual leukocytes migrating in the tailfin of unwounded fish (left panel) or towards the tailfin wound (right panels). No wound, n = 11; control/PKC-ζ-WT, n = 12; control/PKC-ζ-KW, n = 10; control/PAR-6-NT, n = 13; control/PAR-3-aPKCBR, n = 13). Tracks are from one representative experiment of at least three independent experiments. (E–G) Quantification of 2D (E) speed, (F) path straightness and (G) directional speed ratio of myeloid cells during the wound response. Data are expressed as the mean±s.e.m. of at least three separate experiments (PKC-ζ-WT, n = 27 cells in three larvae; PKC-ζ-KW, n = 27 cells in three larvae; PAR-6-NT, n = 45 cells in four larvae; PAR-3-aPKCBR, n = 64 cells in five larvae); *P<0.05; **P<0.01; ***P<0.001; ns, non-significant (two-tailed unpaired Student's t-test). See also supplementary material Fig. S1; Movies 1, 2.
Mentions: To determine how PAR-3, PAR-6 and aPKC regulate the directed migration of leukocytes in vivo in a 3D environment, we developed a model of wound-induced inflammatory cell migration in medaka fish, based on live imaging of tissue-resident myeloid cells expressing membrane-tethered YFP [memYFP, using the transgenic line TG(FmpoP::memYFP) as, in medaka, myeloperoxidase (MPO) is expressed in mixed myeloid lineages that also contain sudanophilic material; supplementary material Fig. S1A] (Aghaallaei et al., 2010; Grabher et al., 2007). Because silencing of the PAR components affects the morphogenesis of several embryonic tissues in zebrafish (Horne-Badovinac et al., 2001; Munson et al., 2008; Wei et al., 2004) and the use of morpholinos is not effective in juvenile medaka (∼9–11 days post-fertilization), we adopted a meganuclease-driven, transient transgenesis approach based on the injection of embryos at the one-cell stage (Rembold et al., 2006). We devised a strategy whereby a set of PAR-complex-interfering mutants, expressed under the myeloid-cell-specific Fmpo promoter (FmpoP), were co-injected with a nuclear-localized fluorescent marker (mCherry fused to histone H2A – H2AmCherry), to track the PAR-mutant-expressing cells (Fig. 1A–C) (Souren et al., 2009). Using this established gene expression system, we first determined that the two transgenes were coexpressed in >72% of cells (supplementary material Fig. S1B–D). As the transgenes were expressed in a mosaic fashion, we could directly compare the control subpopulation (H2AmCherry−) with the PAR-mutant-expressing subset (H2AmCherry+), to assess migration-associated parameters during the wound-response within the same animal (Fig. 1A).

Bottom Line: A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization.However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo.Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy.

Show MeSH
Related in: MedlinePlus