Jocelyn A. McDonald

Par-1 Controls Myosin-II Activity through Myosin Phosphatase to Regulate Border Cell Migration

BACKGROUND Localized actomyosin contraction couples with actin polymerization and cell-matrix adhesion to regulate cell protrusions and retract trailing edges of migrating cells. Although many cells migrate in collective groups during tissue morphogenesis, mechanisms that coordinate actomyosin dynamics in collective cell migration are poorly understood. Migration of Drosophila border cells, a genetically tractable model for collective cell migration, requires nonmuscle myosin-II (Myo-II). How Myo-II specifically controls border cell migration and how Myo-II is itself regulated is largely unknown. RESULTS We show that Myo-II regulates two essential features of border cell migration: (1) initial detachment of the border cell cluster from the follicular epithelium and (2) the dynamics of cellular protrusions. We further demonstrate that the cell polarity protein Par-1 (MARK), a serine-threonine kinase, regulates the localization and activation of Myo-II in border cells. Par-1 binds to myosin phosphatase and phosphorylates it at a known inactivating site. Par-1 thus promotes phosphorylated myosin regulatory light chain, thereby increasing Myo-II activity. Furthermore, Par-1 localizes to and increases active Myo-II at the cluster rear to promote detachment; in the absence of Par-1, spatially distinct active Myo-II is lost. CONCLUSIONS We identify a critical new role for Par-1 kinase: spatiotemporal regulation of Myo-II activity within the border cell cluster through localized inhibition of myosin phosphatase. Polarity proteins such as Par-1, which intrinsically localize, can thus directly modulate the actomyosin dynamics required for border cell detachment and migration. Such a link between polarity proteins and cytoskeletal dynamics may also occur in other collective cell migrations.

On the Role of PDZ Domain-Encoding Genes in Drosophila Border Cell Migration

Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.

Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration.

Background: Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. Results: Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par‐1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E‐Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh‐Rac and Hh‐Par‐1 signaling was illustrated in the wing, in which Hh‐dependent phenotypes were enhanced by loss of Rac or par‐1. Conclusions: We identified a pathway by which Hh signaling connects to Rac and Par‐1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways. Developmental Dynamics 242:414–431, 2013.

Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue

A challenge for migrating collectives is to respond to physical changes in local environments. Border cells migrate collectively in the Drosophila ovary and require dynamic myosin to maintain their morphology. Border cells elevate active myosin in response to tissue compression. Myosin tension counteracts tissue constraints for collective movement.

Canonical and Noncanonical Roles of Par-1/MARK Kinases in Cell Migration

The partitioning defective gene 1 (Par-1)/microtubule affinity-regulating kinase (MARK) family of serine-threonine kinases have diverse cellular roles. Primary among these roles are the establishment and maintenance of cell polarity and the promotion of microtubule dynamics. Par-1/MARK kinases also regulate a growing number of cellular functions via noncanonical protein targets. Recent studies have demonstrated that Par-1/MARK proteins are required for the migration of multiple cell types. This review outlines the current evidence for regulation of cell migration by Par-1/MARK through both canonical and noncanonical roles. Par-1/MARK canonical control of microtubules during nonneuronal and neuronal migration is described. Next, regulation of cell polarity by Par-1/MARK and its dynamic effect on the movement of migrating cells are discussed. As examples of recent research that have expanded, the roles of the Par-1/MARK in cell migration, noncanonical functions of Par-1/MARK in Wnt signaling and actomyosin dynamics are described. This review also highlights questions and current challenges to further understanding how the versatile Par-1/MARK proteins function in cell migration during development, homeostatic processes, and cancer.

Dynamics of cell polarity in tissue morphogenesis: a comparative view from Drosophila and Ciona.

Tissues in developing embryos exhibit complex and dynamic rearrangements that shape forming organs, limbs, and body axes. Directed migration, mediolateral intercalation, lumen formation, and other rearrangements influence the topology and topography of developing tissues. These collective cell behaviors are distinct phenomena but all involve the fine-grained control of cell polarity. Here we review recent findings in the dynamics of polarized cell behavior in both the Drosophila ovarian border cells and the Ciona notochord. These studies reveal the remarkable reorganization of cell polarity during organ formation and underscore conserved mechanisms of developmental cell polarity including the Par/atypical protein kinase C (aPKC) and planar cell polarity pathways. These two very different model systems demonstrate important commonalities but also key differences in how cell polarity is controlled in tissue morphogenesis. Together, these systems raise important, broader questions on how the developmental control of cell polarity contributes to morphogenesis of diverse tissues across the metazoa.

Drosophila Condensin II subunit Chromosome-associated protein D3 regulates cell fate determination through non-cell-autonomous signaling

The pattern of the Drosophila melanogaster adult wing is heavily influenced by the expression of proteins that dictate cell fate decisions between intervein and vein during development. dSRF (Blistered) expression in specific regions of the larval wing disc promotes intervein cell fate, whereas EGFR activity promotes vein cell fate. Here, we report that the chromatin-organizing protein CAP-D3 acts to dampen dSRF levels at the anterior/posterior boundary in the larval wing disc, promoting differentiation of cells into the anterior crossvein. CAP-D3 represses KNOT expression in cells immediately adjacent to the anterior/posterior boundary, thus blocking KNOT-mediated repression of EGFR activity and preventing cell death. Maintenance of EGFR activity in these cells depresses dSRF levels in the neighboring anterior crossvein progenitor cells, allowing them to differentiate into vein cells. These findings uncover a novel transcriptional regulatory network influencing Drosophila wing vein development, and are the first to identify a Condensin II subunit as an important regulator of EGFR activity and cell fate determination in vivo. Summary: The chromatin-organizing protein CAP-D3 maintains EGFR signaling in the developing Drosophila wing disc, identifying a key transcriptional regulatory network influencing vein development.

A biochemical network controlling basal myosin oscillation

The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. Here, we show that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, we uncover an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.The actomyosin cytoskeleton is known to spontaneously oscillate in many systems but the mechanism of this behavior is not clear. Here Qin et al. define a signaling network involving a ROCK-dependent self-activation loop and recruitment of myosin II to the cortex, followed by a local accumulation of myosin phosphatase that shuts off the signal.

The Human dsRNA binding protein PACT is unable to functionally substitute for the Drosophila dsRNA binding protein R2D2

The dsRNA binding protein (dsRBP) PACT was first described as an activator of the dsRNA dependent protein kinase PKR in response to stress signals. Additionally, it has been identified as a component of the small RNA processing pathway. A role for PACT in this pathway represents an important interplay between two modes of post-transcriptional gene regulation. The function of PACT in this context is poorly understood. Thus, additional approaches are required to clarify the mechanism by which PACT functions. In this study, the genetic utility of Drosophila melanogaster was employed to identify dsRNA-binding proteins that are functionally orthologous to PACT. Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis. Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.