Differential phosphorylation of the myosin light chain by multiple kinase pathways is required for collective cell migration. Jocelyn A. McDonald^1,2, Pralay Majumder¹, George Aranjuez^1,2, Ashley Burtscher¹. 1) Molecular Genetics, Cleveland Clinic, Cleveland, OH; 2) Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH.

   Despite the importance of collective cell movement to processes such as organ morphogenesis and tumor invasion and metastasis, how groups of cells escape from epithelia in tissues and subsequently coordinate their motility is still poorly understood. We use the border cell model to uncover novel mechanisms that regulate collective cell migration. Border cells migrate as a cohesive group of 6-10 cells during Drosophila oogenesis, where they detach from the follicular epithelium and migrate 100 m to reach the oocyte. During migration, border cells extend directed protrusions to sense guidance cues and provide traction to their substrate. We discovered that the serine-threonine kinase and cell polarity protein Par-1 is required for efficient detachment of border cells from the epithelium and for protrusion dynamics and directionality. Moreover, we found that a major role of Par-1 in border cells is to specifically regulate the dynamic subcellular localization and activation of myosin-II (myo-II). Par-1 phosphorylates and inactivates myosin phosphatase, thus increasing the levels of active myo-II through enhanced phosphorylation of the myosin regulatory light chain (MRLC/Sqh). Here, we provide evidence that Par-1 integrates with a second kinase, Rho-kinase (Rok), which directly activates myo-II and inactivates the myosin phosphatase. Loss of both Rok and par-1 severely impairs the ability of border cells to detach and migrate. We establish that these two kinases (and additional myo-II activating kinases) differentially regulate mono- versus di-phosphorylation of MRLC, thus resulting in distinct pools of active myo-II. While Rok and myosin phosphatase are distributed uniformly in the border cell cluster, Par-1 localizes to the cluster rear; this gives rise to polarized active myo-II. We propose that Rok, localized Par-1 and other kinases jointly activate myo-II in a spatio-temporally defined manner during the detachment and protrusion extension of border cells.