Abstract
ABSTRACTInflammatory 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.
Highlights
Polarization allows cells to sense and to elicit the proper spatiotemporal responses to cues that arise from the surrounding microenvironment
Using high-resolution live imaging in genetically engineered medaka fish larvae (Oryzias latipes), we show that core components of the PAR complex regulate wound-induced directional migration of myeloid cells, through modulation of ROCK-dependent F-actin and microtubule organizing center (MTOC) dynamics
PAR proteins promote the directed migration of myeloid cells in vivo To determine how PAR-3, PAR-6 and atypical protein kinase C (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 tissueresident 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 contain sudanophilic material; supplementary material Fig. S1A] (Aghaallaei et al, 2010; Grabher et al, 2007)
Summary
Polarization allows cells to sense and to elicit the proper spatiotemporal responses to cues that arise from the surrounding microenvironment. At the molecular level, polarized migration involves the establishment and maintenance of a spatial and functional asymmetry of molecular components between the anterior (leading) and posterior (trailing) edges of the migrating cell (Lauffenburger and Horwitz, 1996). How such coordinate partitioning of the cell migration and signaling machinery is controlled and maintained over time is largely unknown. Received 12 November 2013; Accepted 29 July 2014 models evoke the creation of asymmetry in the distribution of key signaling molecules in the migrating cell, either through the production of rapidly diffusing inhibitory molecules by the front of the cell or through the sequestration of limiting polarity components to the front (Wang, 2009). Studies performed in 2D substrates can be highly detailed but often provide results that differ substantially from those obtained in vivo under 3D conditions (Lammermann et al, 2008; Pouthas et al, 2008)
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