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

ABSTRACT Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. 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. Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae (Oryzias latipes). 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. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

. 85 Movie 1. Effects of PKC-ζ-WT expression on myeloid cell migration to a tailfin wound. Wounding response in 11dpf TG(Fm-poP::memYFP) transgenic larva co-expressing nuclear H2AmCherry and PKC-ζ-WT in memYFP + leukocytes. YFP (green), Cherry (red) images were obtained every 20 s with a spinning-disk confocal microscope. The wound is located on the bottom right hand side of the movie. Frame rate: 15 fps.
Movie 3. Imaging of cytoskeletal dynamics during wound-response. Wounding response in 9dpf TG(FmpoP::EB3-EGFP/Fm-poP::RFP-Lifeact) transgenic larvae expressing nuclear H2B-CFP in myeloid cells. CFP (blue), GFP (green), RFP (red) images were obtained with a spinning-disk confocal microscope. The wound is located on the right hand side of the movie. Individual frames acquired every 16.5 s. Frame rate: 12 fps.

Movie 2. Effects of PKC-ζ-KW expression on myeloid cell migration to a tailfin wound.
Wounding response in 11dpf TG(Fm-poP::memYFP) transgenic larva co-expressing nuclear H2AmCherry and PKC-ζ-KW in memYFP + leukocytes. YFP (green), Cherry (red) images were obtained every 20.8 s with a spinning-disk confocal microscope. The wound is located on the bottom right hand side of the movie. Frame rate: 15 fps.
Movie 4. F-actin dynamics in leukocytes moving to wounds. F-actin dynamics of representative myeloid cells during wound-response. CFP (blue), GFP (green), RFP (red) images were obtained with a spinning-disk confocal microscope. A pseudo-color image of RFP-Lifeact was used to map intensity levels of F-actin. Blue to yellow denote low to high intensities, respectively. Wounds are located on the right hand side of the movie. (A) Individual frames acquired every 16.5 s. (B) Individual frames acquired every 8.4 s. Frame rate: 6 fps.
Movie 6. Effect of PAR mutants on F-actin dynamics. F-actin dynamics of representative wound-activated myeloid cells expressing PKC-ζ-WT or the different PAR-mutants. CFP (blue), GFP (green), RFP (red) images were obtained every 15-18 s with a spinning-disk confocal microscope. A pseudo-color image of RFP-Lifeact was used as in Movie 4. Wounds are located on the right hand side of the movie. Frame rate: 6 fps.
Movie 5. MTOC repositioning in leukocytes moving to wounds. MTOC positioning in the perinuclear region of cells from Movie 4. CFP (magenta), GFP (green) images were obtained with a spinning-disk confocal microscope. A digitalized reconstruction of the MTOC position (green dot) was created to illustrate MTOC orientation around the nucleus (magenta). Frame rate: 6 fps.
Movie 7. Effect of PAR mutants on MTOC mobility. MTOC positioning in the perinuclear region of leukocytes from Movie 6. CFP (magenta), GFP (green) images were obtained every 15-18 s with a spinning-disk confocal microscope. A digitalized reconstruction of the MTOC position (green dot) was created as in Movie 5. Frame rate: 6 fps.
Movie 9. Perturbation of Rho signaling on MTOC dynamics. MTOC positioning in the perinuclear region of leukocytes from Movie 8. CFP (magenta), GFP (green) images were obtained every 15 s with a spinning-disk confocal microscope. A digitalized reconstruction of the MTOC position (green dot) was created as in Movie 5. Frame rate: 6 fps.
Movie 8. Perturbation of Rho signaling on F-actin dynamics. F-actin dynamics of wound-activated myeloid cells treated with Y-27632 (500 µM). CFP (blue), GFP (green), RFP (red) images were obtained every 15 s with a spinning-disk confocal microscope. A pseudo-color image of RFP-Lifeact was used as in Movie 4. Wounds are located on the right hand side of the movie. Frame rate: 6 fps.
Movie 10. Effect of Rho-kinase inhibition on GFP-PKC-ζ polarity. GFP-PKC-ζ polarization in representative myeloid cells migrating to a wound in control or Y-27632-treated TG(FmpoP::mCherry) transgenic larvae, at 9dpf. GFP (green) and Cherry (magenta) images were obtained every 15 s with a spinning-disk confocal microscope. Ratio images of GFP/Cherry were generated to reveal increases in GFP-PKC-ζ fluorescence intensities. Blue to red colors denote low to high ratio values, respectively. Wounds are located on the right hand side of the movie. Frame rate: 5 fps.
Movie 12. Effect of PKC-ζ enzymatic function on RhoA activity. RhoA activity in migrating myeloid cells co-expressing the cytosolic RhoA-FRET biosensor and PKC-ζ-WT or PKC-ζ-KW in 9dpf TG(FmpoP::mCherry) transgenic larvae. Ratio images of YFP/CFP are shown to reveal the average activation level of RhoA. Blue to white denote low to high ratio values, respectively. Wounds are located on the right hand side of the movie. Frame rate: 4 fps.
Movie 11. Perturbation of PKC-ζ activity on GFP-PKC-ζ "front" polarity. GFP-PKC-ζ polarization in representative wound-activated myeloid cells expressing PKC-ζ-WT or PKC-ζ-KW in 9dpf TG(FmpoP::mCherry) transgenic larvae. GFP (green) and Cherry (magenta) images were obtained every 15 s with a spinning-disk confocal microscope. Ratio images of GFP/Cherry were generated as in Movie 10. Wounds are located on the right hand side of the movie. Frame rate: 5 fps. In the Rayleigh test for uniformity of directional data, the degree of deviation from random is given (Mardia and Jupp, 2000). Results of the Rayleigh test are displayed on a scale from random (-) to not at all random (+). In the Watson two-sample test for homogeneity of two samples of circular data, the higher the p-value, the more similar the populations (Mardia and Jupp, 2000).