Summary Cell polarity is crucial for directed migration. Here we show that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P3-PI(3,4)P2 during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism.

SummaryTissue wounding induces the rapid recruitment of leukocytes1. Wounds and tumors, a type of “unhealed wound”2, generate hydrogen peroxide (H2O2) through a NADPH oxidase (NOX) and the extracellular H2O2 mediates recruitment of leukocytes, particularly first responders of innate immunity, neutrophils, to injured tissue3–6. However, it is not known what sensor neutrophils use to detect the redox state at wounds. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H2O2 following tissue injury and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H2O2-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signaling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

Summary Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Non-invasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling in vivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively-active CXCR4 mutation in zebrafish WHIM syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.