Zhang Y, Kim TH, Niswander L. Phactr4 regulates directional migration of enteric neural crest through PP1, integrin signaling, and cofilin activity. Genes Dev 26: 69-81

Howard Hughes Medical Institute, Department of Pediatrics, Graduate Program in Cell Biology, Stem Cells, and Development, Children's Hospital Colorado, University of Colorado, Aurora, Colorado 80045, USA.
Genes & development (Impact Factor: 10.8). 01/2012; 26(1):69-81. DOI: 10.1101/gad.179283.111
Source: PubMed


Hirschsprung disease (HSCR) is caused by a reduction of enteric neural crest cells (ENCCs) in the gut and gastrointestinal blockage. Knowledge of the genetics underlying HSCR is incomplete, particularly genes that control cellular behaviors of ENCC migration. Here we report a novel regulator of ENCC migration in mice. Disruption of the Phactr4 gene causes an embryonic gastrointestinal defect due to colon hypoganglionosis, which resembles human HSCR. Time-lapse imaging of ENCCs within the embryonic gut demonstrates a collective cell migration defect. Mutant ENCCs show undirected cellular protrusions and disrupted directional and chain migration. Phactr4 acts cell-autonomously in ENCCs and colocalizes with integrin and cofilin at cell protrusions. Mechanistically, we show that Phactr4 negatively regulates integrin signaling through the RHO/ROCK pathway and coordinates protein phosphatase 1 (PP1) with cofilin activity to regulate cytoskeletal dynamics. Strikingly, lamellipodia formation and in vivo ENCC chain migration defects are rescued by inhibition of ROCK or integrin function. Our results demonstrate a previously unknown pathway in ENCC collective migration in vivo and provide new candidate genes for human genetic studies of HSCR.

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    • "Studies of ENCC migration to date have focused on the caudal advance of the ENCC wavefront [21-27]. ENCCs migrate in chains with high cell-cell contact [21,23,24,28], and so little is known about how individual ENCCs behave to ensure that all regions of the gut are also evenly populated with ENCCs. We had previously assumed that each gut region is colonized by sub-populations of ENCCs stopping as the wavefront of ENCCs moves caudally [23]. "
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    ABSTRACT: Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analysed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region. The behaviour of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole. Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.
    BMC Biology 03/2014; 12(1):23. DOI:10.1186/1741-7007-12-23 · 7.98 Impact Factor
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    • "NC cells crawl through a variety of extracellular matrix (ECM) including regions rich in fibronectin and laminin (Newgreen and Thiery, 1980; Strachan and Condic, 2003; Brauer and Markwald, 1987). Evidence from chick and mouse time-lapse imaging has revealed that NC cell chain migration occurs in the cranial, trunk and intestinal subregions of the embryo (Kulesa and Fraser 1998; Young, Anderson et al. 2004; Kasemeier-Kulesa, Bradley et al. 2006; Druckenbrod and Epstein 2007; Rupp and Kulesa 2007; Nishiyama, Uesaka et al. 2012; Zhang, Kim et al. 2012). NC cells in nearly every vertebrate model system (chick, mouse, zebrafish, axolotl, turtle, snake) have also been observed to travel in multicellular streams (Reyes et al., 2010; Kulesa and Fraser, 1998; Schilling and Kimmel, 1994; Golding et al., 2000; Epperlein et al., 2007; Gilbert et al., 2007). "
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    ABSTRACT: Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.
    Physical Biology 06/2013; 10(3):035003. DOI:10.1088/1478-3975/10/3/035003 · 2.54 Impact Factor
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    • "Cofilin and Phactr4 have also been shown to colocalize at the edge of the lamellipodium of migrating mouse embryonic fibroblasts (Zhang et al., 2012). Defects in the regulation of the actin monomer pool may therefore also explain the findings that many Phactr family members seem to be required for cell motility (Sagara et al., 2009; Zhang et al., 2012). "
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    ABSTRACT: Phactr proteins bind actin and protein phosphatase 1 (PP1), and are involved in processes ranging from angiogenesis to cell cycle regulation. Phactrs share a highly conserved RPEL domain with the Myocardin Related Transcription Factor (MRTF) family, where actin-binding to this domain regulates both the nuclear localization and the activity of these transcription coactivators. We show here that in contrast to MRTF-A, the RPEL domain is dispensable for the subcellular localization of Phactr4. Instead, we find the domain facilitating competitive binding of monomeric actin and PP1 to Phactr4. Binding of actin to Phactr4 influences the activity of PP1 and the phosphorylation status of one of its downstream targets, cofilin. Consequently, at low actin monomer levels, Phactr4 guides PP1 to dephosphorylate cofilin. This active form of cofilin is then able to sever and depolymerise actin filaments and thus restore the actin monomer pool. Accordingly, our data discloses the central role of Phactr4 in a feedback loop, where actin monomers regulate their own number via the activation of a key regulator of actin dynamics. Depending on the protein context, the RPEL domain can thus elicit mechanistically different responses to maintain the cellular actin balance.
    Journal of Cell Science 11/2012; 126(2). DOI:10.1242/jcs.113241 · 5.43 Impact Factor
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