Guide through the blizzard

AbstractHenry Bourne finds a new book a thoughtful and knowledgeable travel guide to the key principles behind the bewildering variety of interactions regulating expression of specific genes

Q & A

AbstractHenry Bourne is Professor of Cellular and Molecular Pharmacology and Medicine at the University of California, San Francisco (UCSF). For many years his research focused on trimeric G proteins and G protein coupled receptors. Five years ago, he turned to studying the ‘compass’ used by blood cells to interpret gradients of chemical attractants and migrate to sites where they are needed to defend the host. He has been elected to membership in the American Academy of Arts and Sciences and the National Academy of Sciences, USA

PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells

Chemoattractants such as formyl-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent pathways that promote formation of a protrusive front and contracting back and sides. RhoA, a Rho GTPase, stimulates assembly of actomyosin contractile complexes at the sides and back. We show here, in differentiated HL60 cells, that PDZRhoGEF (PRG), a guanine nucleotide exchange factor (GEF) for RhoA, mediates RhoA-dependent responses and determines their spatial distribution. As with RNAi knock-down of PRG, a GEF-deleted PRG mutant blocks fMLP-dependent RhoA activation and causes neutrophils to exhibit multiple fronts and long tails. Similarly, inhibition of RhoA, a Rho-dependent protein kinase (ROCK), or myosin II produces the same morphologies. PRG inhibition reduces or mislocalizes monophosphorylated myosin light chains in fMLP-stimulated cells, and myosin II ATPase inhibition reciprocally disrupts normal localization of PRG. We propose a cooperative reinforcing mechanism at the back of cells, in which PRG, RhoA, ROCK, myosin II, and actomyosin spatially cooperate to consolidate attractant-induced contractility and ensure robust cell polarity

Characterisation of platelet receptor CLEC-2 in thrombosis and inflammation

Platelets have an established role in thrombosis and haemostasis. C-type lectin-like receptor 2 (CLEC-2) is highly expressed on platelets and a subpopulation of myeloid cells, and is critical in lymphatic development. Using a recently developed mouse model for platelet-specific deletion of platelet-CLEC-2, CLEC1bfl/flGPIb-Cre, we show that CLEC-2 contributes to thrombus stability in mice. However, CLEC-2 inhibition in human blood using monoclonal antibody, AYP1, or recombinant CLEC-2 did not translate these observations to human. This presents CLEC-2 as a promising therapeutic target in thromboinflammatory or inflammatory disease, without detriment to thrombosis and haemostasis. We show that hemin, the oxidised form of haem released post-haemolysis, is a novel CLEC-2 ligand which can activate and aggregate platelets through its hemITAM domain. Interestingly, anti-malarial drug hydroxychloroquine is able to inhibit hemin-induced mouse and human platelet activation, but not ROS generation. In a mouse model of thrombosis, the ferric chloride injury of the carotid artery, we demonstrate that hydroxychloroquine treatment presents small, non-occlusive thrombi, compared to saline-treated controls. This may present hemin-CLEC-2 as a promising therapeutic target in thromboinflammatory disease. Beyond thrombosis, platelets contribute to the development, progression and resolution of the inflammatory response. CLEC-2 is protective during mouse models of sepsis, and other inflammatory diseases. Here we show that CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from inflamed tissue during murine peritonitis through its ligand, podoplanin. Treatment using recombinant CLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. Both the thromboinflammatory and immunomodulatory functions of CLEC-2 present strategies to reduce tissue inflammation and inflammatory thrombi, and could be therapeutically exploited to limit disease progression

Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42

William E. Barry Correspondence

Entries include typed letters on personal stationery from Bourne presenting books to the Maine Author Collection, and specifically a book published by his executors, as a provision from Barry\u27s wil

Signal Processing

Contains research objectives, summary of research and reports on two research projects.Joint Services Electronics Programs (U. S. Army, U. S. Navy, and U. S. Air Force) under Contract DAAB07-71-C-0300U. S. Coast Guard (Contract DOT-CG-13446-A

Signal Processing

Contains research objectives and reports on two research projects.Joint Services Electronics Programs (U. S. Army, U. S. Navy, and U. S. Air Force) under Contract DA 28-043-AMC-02536(E