The PAR complex controls the spatiotemporal dynamics of F-actin and the MTOC in directionally migrating leukocytes.
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.
Affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy.Show MeSH
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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).
Affiliation: Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, 20132 Milan, Italy.