Lost Dollars Threaten Research in Public Academic Health Centers.

The decrease of federal and state support threatens long-term sustainability of research in publicly supported academic health centers. In weathering these financial threats, research at the University of California, San Francisco (UCSF), has undergone 3 substantial changes: institutional salary support goes preferentially to senior faculty, whereas the young increasingly depend on grants; private and government support for research grows apace in clinical departments but declines in basic science departments; and research is judged more on its quantity (numbers of investigators and federal and private dollars) than on its goals, achievements, or scientific quality. We propose specific measures to alleviate these problems. Other large public academic health centers probably confront similar issues, but-except for UCSF-such centers have not been subjected to detailed public analysis.-Bourne, H. R., Vermillion, E. B. Lost dollars threaten research in public academic health centers.

A fair deal for PhD students and postdocs.

The relentless expansion that threatens the sustainability of biomedical research in the US takes a heavy toll on young researchers.

A recipe for mediocrity and disaster, in five axioms.

Biomedical research in the US will become unsustainable unless scientists and research institutions start to question certain assumptions they have long taken for granted.

The writing on the wall.

The biomedical research enterprise in the US has become unsustainable and urgent action is needed to address a variety of problems, including a lack of innovation, an over-reliance on soft money for faculty salaries, the use of graduate students as a source of cheap labour, and a 'holding tank' full of talented postdocs with limited career opportunities.

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.

Bound attractant at the leading vs. the trailing edge determines chemotactic prowess.

We have analyzed chemotaxis of neutrophil-differentiated HL60 cells in microfluidic devices that create exponential gradients of the chemoattractant, f-Met-Leu-Phe (fMLP). Such gradients expose each cell to a difference in fMLP concentration (DeltaC) across its diameter that is directly proportional to the ambient concentration (C) at that cell's position in the gradient, so the ratio DeltaC/C is constant everywhere. Cells exposed to ambient fMLP concentrations near the constant of dissociation (K(d)) for fMLP binding to its receptor ( approximately 10 nM) crawl much less frequently when DeltaC/C is 0.05 than when it is 0.09 or 0.13. Hence, cells can detect the gradient across their diameter without moving and, thus, without experiencing temporal changes in attractant concentration. At all DeltaC/C ratios tested, the average chemotactic prowess of individual cells (indicated by the distance a cell traveled in the correct direction divided by the length of its migration path) is maximal for cells that start migrating at concentrations near the K(d) and progressively decreases at higher or lower starting concentrations.

Polarity reveals intrinsic cell chirality.

Like blood neutrophils, dHL60 cells respond to a uniform concentration of attractant by polarizing in apparently random directions. How each cell chooses its own direction is unknown. We now find that an arrow drawn from the center of the nucleus of an unpolarized cell to its centrosome strongly predicts the subsequent direction of attractant-induced polarity: Of 60 cells that polarized in response to uniform f-Met-Leu-Phe (fMLP), 42 polarized to the left of this arrow, 6 polarized to the right, and 12 polarized directly toward or away from the centrosome. To investigate this directional bias we perturbed a regulatory pathway, downstream of Cdc42 and partitioning-defective 6 (Par6), which controls centrosome orientation relative to polarity of other cells. Dominant negative Par6 mutants block polarity altogether, as previously shown for disrupting Cdc42 activity. Cells remain able to polarize, but without directional bias, if their microtubules are disrupted with nocodazole, or they express mutant proteins that interfere with activities of PKCzeta or dynein. Expressing constitutively active glycogen synthase kinase 3beta (GSK3beta) causes cells to polarize preferentially to the right. Distributions of most of these polarity regulators localize to the centrosome but show no left-right asymmetry before polarization. Together, these findings suggest that an intrinsically chiral structure, perhaps the centrosome, serves as a template for directing polarity in the absence of spatial cues. Such a template could help to determine left-right asymmetry and planar polarity in development.

Morphology matters in immune cell chemotaxis: membrane asymmetry affects amplification.

A key mediator of eukaryotic chemotaxis is the asymmetric accumulation of phosphatidylinositol-3,4,5-triphosphate (PIP3) on the cell membrane. Recent work has focused on understanding how a shallow external gradient of chemoattractant leads to an amplified internal gradient of PIP3. In this paper we dissect what fraction of this amplification is derived biochemically by the signal transduction network and how much arises entirely from the effects of cell morphology. Here we identify and formalize the role of morphology in signal detection and demonstrate its effects through simulation and experiments. Our key result is that an asymmetric distribution of membrane accounts for approximately one-half of the measured amplification from ligand concentration to PIP3 production. We also show that the underlying biochemical network behaves as a linear amplifier in the micropipette assay.

Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.

Wound healing is essential for maintaining the integrity of multicellular organisms. In every species studied, disruption of an epithelial layer instantaneously generates endogenous electric fields, which have been proposed to be important in wound healing. The identity of signalling pathways that guide both cell migration to electric cues and electric-field-induced wound healing have not been elucidated at a genetic level. Here we show that electric fields, of a strength equal to those detected endogenously, direct cell migration during wound healing as a prime directional cue. Manipulation of endogenous wound electric fields affects wound healing in vivo. Electric stimulation triggers activation of Src and inositol-phospholipid signalling, which polarizes in the direction of cell migration. Notably, genetic disruption of phosphatidylinositol-3-OH kinase-gamma (PI(3)Kgamma) decreases electric-field-induced signalling and abolishes directed movements of healing epithelium in response to electric signals. Deletion of the tumour suppressor phosphatase and tensin homolog (PTEN) enhances signalling and electrotactic responses. These data identify genes essential for electrical-signal-induced wound healing and show that PI(3)Kgamma and PTEN control electrotaxis.

To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front.

Chemoattractants like f-Met-Leu-Phe (fMLP) induce neutrophils to polarize by triggering divergent signals that promote the formation of protrusive filamentous actin (F-actin; frontness) and RhoA-dependent actomyosin contraction (backness). Frontness locally inhibits backness and vice versa. In neutrophil-like HL60 cells, blocking phosphatidylinositol-3,4,5-tris-phosphate (PIP3) accumulation with selective inhibitors of PIP3 synthesis completely prevents fMLP from activating a PIP3-dependent kinase and Cdc42 but not from stimulating F-actin accumulation. PIP3-deficient cells show reduced fMLP-dependent Rac activity and unstable pseudopods, which is consistent with the established role of PIP3 as a mediator of positive feedback pathways that augment Rac activation at the front. Surprisingly, such cells also show reduced RhoA activation and RhoA-dependent contraction at the trailing edge, leading to the formation of multiple lateral pseudopods. Cdc42 mediates PIP3's positive effect on RhoA activity. Thus, PIP3 and Cdc42 maintain stable polarity with a single front and a single back not only by strengthening pseudopods but also, at longer range, by promoting RhoA-dependent actomyosin contraction at the trailing edge.

Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis.

Migrating cells need to make different actin assemblies at the cell's leading and trailing edges and to maintain physical separation of signals for these assemblies. This asymmetric control of activities represents one important form of cell polarity. There are significant gaps in our understanding of the components involved in generating and maintaining polarity during chemotaxis. Here we characterize a family of complexes (which we term leading edge complexes), scaffolded by hematopoietic protein 1 (Hem-1), that organize the neutrophil's leading edge. The Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE)2 complex, which mediates activation of actin polymerization by Rac, is only one member of this family. A subset of these leading edge complexes are biochemically separable from the WAVE2 complex and contain a diverse set of potential polarity-regulating proteins. RNA interference-mediated knockdown of Hem-1-containing complexes in neutrophil-like cells: (a) dramatically impairs attractant-induced actin polymerization, polarity, and chemotaxis; (b) substantially weakens Rac activation and phosphatidylinositol-(3,4,5)-tris-phosphate production, disrupting the (phosphatidylinositol-(3,4,5)-tris-phosphate)/Rac/F-actin-mediated feedback circuit that organizes the leading edge; and (c) prevents exclusion of activated myosin from the leading edge, perhaps by misregulating leading edge complexes that contain inhibitors of the Rho-actomyosin pathway. Taken together, these observations show that versatile Hem-1-containing complexes coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving WAVE2-dependent actin polymerization.

Neutrophil microtubules suppress polarity and enhance directional migration.

How do microtubules, which maintain and direct polarity of many eukaryotic cells, regulate polarity of blood neutrophils? In sharp contrast to most cells, disrupting a neutrophil's microtubule network with nocodazole causes it to polarize and migrate [Niggli, V. (2003) J. Cell Sci. 116, 813-822]. Nocodazole induces the same responses in differentiated HL-60 cells, a model neutrophil cell line, and reduces their chemotactic prowess by causing them to pursue abnormally circuitous paths in migrating toward a stationary point source of an attractant, f-Met-Leu-Phe (fMLP). The chemotactic defect stems from dramatic nocodazole-induced imbalance between the divergent, opposed fMLP-induced "backness" and "frontness" signals responsible for neutrophil polarity. Nocodazole (i) stimulates backness by increasing Rho- and actomyosin-dependent contractility, as reported by Niggli, and also (ii) impairs fMLP-dependent frontness: pseudopods are flatter, contain less F-actin, and show decreased membrane translocation of PH-Akt-GFP, a fluorescent marker for 3'-phosphoinositide lipids. Inhibiting backness with a pharmacologic inhibitor of a Rho-dependent kinase substantially reverses nocodazole's effects on chemotaxis, straightness of migration paths, morphology, and PH-Akt-GFP translocation. Thus, microtubules normally balance backness vs. frontness signals, preventing backness from reducing the strength of pseudopods and from impairing directional migration.

Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils.

Like neutrophilic leukocytes, differentiated HL-60 cells respond to chemoattractant by adopting a polarized morphology, with F-actin in a protruding pseudopod at the leading edge and contractile actin-myosin complexes at the back and sides. Experiments with pharmacological inhibitors, toxins, and mutant proteins show that this polarity depends on divergent, opposing "frontness" and "backness" signals generated by different receptor-activated trimeric G proteins. Frontness depends upon Gi-mediated production of 3'-phosphoinositol lipids (PI3Ps), the activated form of Rac, a small GTPase, and F-actin. G12 and G13 trigger backness signals, including activation of a second GTPase (Rho), a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back. The latter effect explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn.

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.