Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach

Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.     

Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation

A hallmark of the response to high-dose radiation is the up-regulation and phosphorylation of proteins involved in cell cycle checkpoint control, DNA damage signaling, DNA repair, and apoptosis. Exposure of cells to low doses of radiation has well documented biological effects, but the underlying regulatory mechanisms are still poorly understood. The objective of this study is to provide an initial profile of the normal human skin fibroblast (HSF) phosphoproteome and explore potential differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques including Trizol extraction of proteins, methylation of tryptic peptides, enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome. Among 494 unique phosphopeptides, 232 were singly phosphorylated, while 262 peptides had multiple phosphorylation sites indicating the overall effectiveness of the IMAC technique to enrich both singly and multiply phosphorylated peptides. We observed approximately 1.9-fold and approximately 3.6-fold increases in the number of identified phosphopeptides in low-dose and high-dose samples respectively, suggesting both radiation levels stimulate cell signaling pathways. A 6-fold increase in the phosphorylation of cyclin dependent kinase (cdk) motifs was observed after low- dose irradiation, while high-dose irradiation stimulated phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT/RSK motifs 8.5- and 5.5-fold, respectively. High- dose radiation resulted in the increased phosphorylation of proteins involved in cell signaling pathways as well as apoptosis while low-dose and control phosphoproteins were broadly distributed among biological processes.     

High-throughput phosphotyrosine profiling using SH2 domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.     

Profiling the Trypanosoma cruzi phosphoproteome

Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here.     

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.     

Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio)

The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease‐causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well‐known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS‐based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif‐X analysis, for 80% of the identified phosphorylation sites, with the proline‐directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos.     

Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean

To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg of total peptides using a combined strategy of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and mass spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate. We identified a total of 2,001 phosphopeptides containing 1,026 unambiguous phosphorylation sites from 956 proteins, with an average false discovery rate of 0.78% for the entire study. The entire data set was uploaded into the Plant Protein Phosphorylation Database (www.p3db.org), including all meta-data and annotated spectra. The Plant Protein Phosphorylation Database is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally determined phosphosites. Comparisons with other large-scale phosphoproteomic studies determined that 652 of the phosphoproteins are novel to this study. The unique proteins fall into several Gene Ontology categories, some of which are overrepresented in our study as well as other large-scale phosphoproteomic studies, including metabolic process and RNA binding; other categories are only overrepresented in our study, like embryonic development. This investigation shows the importance of analyzing multiple plants and plant organs to comprehensively map the complete plant phosphoproteome.     

Depletion of Acidic Phosphopeptides by SAX To Improve the Coverage for the Detection of Basophilic Kinase Substrates

The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.     

Proteome analysis of a single zebrafish embryo using three different digestion strategies coupled with liquid chromatography-tandem mass spectrometry

Zebrafish is a powerful model to analyze vertebrate embryogenesis and organ development. Although a number of genes have been identified to specify embryonic development processes, only a few large-scale proteomic analyses have been reported in regard to these events to date. Here the total proteins of a single embryo were analyzed by urea-, sodium deoxycholate (SDC)-, and performic acid (PA)-assisted trypsin digestion strategies coupled to capillary liquid chromatography-tandem mass spectrometry (CapLC-MS/MS) identification. In total, 509 and 210 proteins were detected from the embryos at 72 and 120 hours postfertilization (hpf), respectively, with a false identification rate of less than 1%. Approximately 95% of those proteins could be observed by combining the urea- and SDC-assisted digestion strategies, suggesting that these two methods are more effective than the PA-assisted method. Compared with 0.5% SDC, 1% SDC was more effective to identify proteins in zebrafish embryos. In addition, removal of the predominant yolk proteins could significantly improve protein identification efficiency. Our study represents the first overview of the protein expression profile of a single zebrafish embryo at 72 or 120 hpf. More important, this single individual proteome methodology could be applied to multiple development stages of wide-type or mutant embryos, providing a simple and powerful way to further our understanding of embryonic development.     

Motif-specific sampling of phosphoproteomes

Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.     

Identification of major ERK-related phosphorylation sites in Gab1

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.     

Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern

In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.     

Dynamic Phosphoproteome Profiling of Zebrafish Embryonic Fibroblasts during Cold Acclimation

Temperature affects almost all aspects of the fish life. To cope with low temperature, fish have evolved the ability of cold acclimation for survival. However, intracellular signaling events underlying cold acclimation in fish remain largely unknown. Here, the formation of cold acclimation in zebrafish embryonic fibroblasts (ZF4) is monitored and the phosphorylation events during the process are investigated through a large‐scale quantitative phosphoproteomic approach. In total, 11 474 phosphorylation sites are identified on 4066 proteins and quantified 5772 phosphosites on 2519 proteins. Serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation accounted for 85.5%, 13.3%, and 1.2% of total phosphosites, respectively. Among all phosphosites, 702 phosphosites on 510 proteins show differential regulation during cold acclimation of ZF4 cells. These phosphosites are divided into six clusters according to their dynamic changes during cold exposure. Kinase–substrate prediction reveals that mitogen‐activated protein kinase (MAPK) among the kinase groups is predominantly responsible for phosphorylation of these phosphosites. The differentially regulated phosphoproteins are functionally associated with various cellular processes such as regulation of actin cytoskeleton and MAPK signaling pathway. These data enrich the database of protein phosphorylation sites in zebrafish and provide key clues for the elucidation of intracellular signaling networks during cold acclimation of fish.     

Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos

Considerable embryonic loss occurs between Gestation Days 15 and 18 in cattle when critical cellular and molecular events occur, including maternal recognition of pregnancy. To gain insight into these events, mRNA differential display analysis was used to identify eight unique cDNA fragments present in greater abundance in 17.5-day than in 15.5-day bovine embryos. Four cDNA fragments, confirmed to be upregulated in 17.5-day embryos using Northern analysis, were cloned and sequenced. Three cDNA fragments shared sequence identities with known homologs: human allograft inflammatory factor-1 (AIF-1), human LERK-5, and bovine interferon-tau. One novel cDNA fragment did not share sequence identity to previously reported genes, except for a similar DNA sequence in the human genome. AIF-1 mRNA was present in developing placenta through Gestation Day 36, and abundant levels were observed in adult bovine spleen and lung. The novel gene, which we have named periattachment factor (PAF), was not detected in adult tissues using Northern analysis or in conceptuses between Days 30 and 36 of pregnancy. Additional sequence information for bPAF was obtained from a cDNA library constructed from a 25-day bovine embryo. The protein corresponding to the open reading frame has four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, a nuclear targeting sequence, but no obvious DNA or RNA binding motifs. Abundant expression of this gene during a narrow but critical window of embryonic development makes it worthy of further study.     

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.