JCB_201409001 1..14

Cell migration is important for many biological processes, including development, immunity, and regeneration. To be persistently motile, cells must first polarize to form a single front and rear. Thus, for actin-based motility, the question of how cells establish that single region of actin polymerization and prevent the formation of secondary fronts has been of great interest. Previous work has largely focused on the role of the small GTPase Rho and its effectors Rho kinase (ROCK) and myosin II. For example, Rho, ROCK, and myosin II inhibition in rapidly motile amoeboid cell types such as Dictyostelium, neutrophils, or monocytes results in the conversion of well-polarized single-front cells into cells with multiple competing protrusions (Wessels et al., 1988; Worthylake and Burridge, 2003; Xu et al., 2003), whereas expression of constitutively active Rho or myosin II results in round stationary cells with no protrusions (Xu et al., 2003). Similarly, in slowly moving epithelial and mesenchymal cells, Rho pathway activation decreases protrusion number and vice versa (Wang et al., 2003; Vicente-Manzanares et al., 2007, 2008, 2011; Mseka and Cramer, 2011). There are several proposed mechanisms for how Rho pathway activation inhibits the formation of multiple protrusions. One possibility is that Rho signaling through ROCK directly inhibits the signaling factors that promote actin polymerization (Li et al., 2005; Ohta et al., 2006). Alternatively, downstream activation of myosin II could promote the conversion of branched actin structures to bundles, which cannot accommodate Arp2/3-based branching, thereby locally inhibiting protrusion formation (VicenteManzanares et al., 2011). Recently, multiple studies have shown that membrane tension can also be involved in restricting cells to a single front (Keren et al., 2008; Houk et al., 2012; Lieber et al., 2013). Specifically, artificially increasing tension causes neutrophils to cease protruding (Houk et al., 2012), whereas decreasing tension can induce polarized keratocytes to form multiple lamellipodia (Lieber et al., 2013). In this model, once a single protrusion is formed, the force of actin polymerization pushing on the membrane increases the membrane tension globally, which then mechanically inhibits polymerization elsewhere. However, the Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fanshaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase