Projecting or moving up a chemical gradient is a universal behavior of living organisms. We tested the ability of S. cerevisiae a-cells to sense and respond to spatial gradients of the mating pheromone α-factor produced in a microfluidics chamber; the focus was on bar1Δ strains, which do not degrade the pheromone input. The yeast cells exhibited good accuracy with the mating projection typically pointing in the correct direction up the gradient (∼80% under certain conditions), excellent sensitivity to shallow gradients, and broad dynamic range so that gradient-sensing was relatively robust over a 1000-fold range of average α-factor concentrations. Optimal directional sensing occurred at lower concentrations (5 nM) close to the Kd of the receptor and with steeper gradient slopes. Pheromone supersensitive mutations (sst2Δ and ste2300Δ) that disrupt the down-regulation of heterotrimeric G-protein signaling caused defects in both sensing and response. Interestingly, yeast cells employed adaptive mechanisms to increase the robustness of the process including filamentous growth (i.e. directional distal budding) up the gradient at low pheromone concentrations, bending of the projection to be more aligned with the gradient, and forming a more accurate second projection when the first projection was in the wrong direction. Finally, the cells were able to amplify a shallow external gradient signal of α-factor to produce a dramatic polarization of signaling proteins at the front of the cell. Mathematical modeling revealed insights into the mechanism of this amplification and how the supersensitive mutants can disrupt accurate polarization. Together, these data help to specify and elucidate the abilities of yeast cells to sense and respond to spatial gradients of pheromone.

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

IntroductionAssembly of membrane signaling complexes in lymphocytes is directed in part by the phospholipid products of phosphoinositide 3-kinase (PI3K) enzymes that are activated following receptor engagement. 1 In T cells, antigen recognition is followed by rapid and sustained accumulation of the PI3K product phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) at the plasma membrane, with particular concentration at the immunologic synapse. [2][3][4][5] The class IA enzymes are thought to be the main subgroup that produces PIP 3 and mediates signals downstream of antigen receptors and costimulatory receptors. 1 Genetic manipulations that enhance PI3K pathway activity cause lymphoproliferation in mice. [6][7][8][9] Conversely, pharmacologic inhibitors of PI3K, such as wortmannin and LY294002, potently block T-and B-cell proliferation. [10][11][12][13] These observations have supported an essential role for PI3K signaling in lymphocyte activation. 1 The clearest link between T lymphocyte signaling and PI3K activation thus far has been through the costimulatory molecule CD28. Phosphorylation of its YXXM motif is thought to be a key means to recruit PI3K enzymes to the cell membrane, and the function of primary T cells is impaired by mutation of this motif. [14][15][16] PI3K enzymes constitute a multigene family, and most members of this family are ubiquitously expressed and comparably sensitive to inhibition by wortmannin and LY294002. 17,18 In addition, wortmannin and LY294002 inhibit other cellular enzymes, including the kinase mTOR that is essential for T-cell proliferation. [18][19][20] Therefore, a precise understanding of PI3K signaling in T cells requires examination of the roles of individual isoforms and subgroups. The 3 class IA catalytic isoforms (p110␣, p110␤, p110␦) exist as heterodimers with 1 of 5 regulatory subunits (p85␣, p55␣, p50␣, p85␤, or p55␥), each possessing conserved Src homology-2 (SH2) domains and other modular domains thought to mediate association with signaling complexes. Class IA regulatory isoforms are essential for stability and localization of the catalytic subunits but possess additional adapter functions independent of their role in regulating class IA PI3K catalytic subunits. 21 Mouse gene-targeting experiments have identified essential functions for p85␣ in B cells and mast cells. 11,[22][23][24] However, T-cell development and function are unimpaired in mice lacking either p85␣, p85␣/ p55␣/p50␣, or p85␤. 11,24,25 Mice lacking p85␣ have impaired T-helper differentiation, but this appears to be due to T-cellextrinsic defects. 22,26 p85␤-deficient T cells show no differences in PI3K signaling responses but have enhanced survival following suboptimal stimulation, suggesting a possible adapter function for p85␤ in a T-cell survival pathway. 25 T cells express all 3 class IA PI3K isoforms (p110␣, p110␤, and p110␦). T cells lacking p110␣ or p110␤ have not been studied, owing to early embryonic lethality in the gene-targeted mice. 27,28 Mice with a knock-in point mutation in p110␦ tha...

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization-or contractilitydriven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.cell migration | mechanosensing | fluorescence polarization microscopy

Photodynamic therapy simultaneously reduces plaque inflammation and promotes repopulation of plaques with a SMC-rich stable plaque cell phenotype while reducing disease progression. These early healing responses suggest that PDT is a promising therapy for the treatment of acute coronary syndromes.

Summary We use super-resolution interferometric photoactivation and localization microscopy (iPALM) and a constrained photoactivatable fluorescent protein integrin fusion to measure the displacement of the head of integrin lymphocyte function-associated 1 (LFA-1) resulting from integrin conformational change on the cell surface. We demonstrate that the distance of the LFA-1 head increases substantially between basal and ligand-engaged conformations, which can only be explained at the molecular level by integrin extension. We further demonstrate that one class of integrin antagonist maintains the bent conformation, while another antagonist class induces extension. Our molecular scale measurements on cell-surface LFA-1 are in excellent agreement with distances derived from crystallographic and electron microscopy structures of bent and extended integrins. Our distance measurements are also in excellent agreement with a previous model of LFA-1 bound to ICAM-1 derived from the orientation of LFA-1 on the cell surface measured using fluorescence polarization microscopy.

Phosphoinositide 3-kinase (PI3K) is a ubiquitously expressed signaling enzyme that plays an integral role in development and activation of B cells. B cell receptor (BCR)-driven proliferation is completely blocked either in cells lacking the p85a regulatory isoform of PI3K or in wild-type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85a to early signaling events has not been fully investigated. Here we show that B cells lacking p85a have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85a results in partial reductions in Ca 2+ mobilization and IjB phosphorylation, whereas ERK phosphorylation is not diminished. Moreover, although Akt phosphorylation is partially reduced, phosphorylation of several proteins downstream of Akt is preserved. These partial impairments suggest that there are other routes to PI3K activation in B cells apart from p85a-associated catalytic subunits. Notably, addition of phorbol ester restores BCR-mediated proliferation in p85a-deficient cells but not wild-type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85a is a failure to activate diacylglycerolregulated signaling enzymes, most likely protein kinase C.See accompanying article: http://dx

Integrins are transmembrane receptors that, upon activation, bind extracellular matrix (ECM) or cell surface ligands and link them to the actin cytoskeleton to mediate cell adhesion and migration1,2. One model for the structural transitions mediating integrin activation termed “the cytoskeletal force hypothesis” posits that force transmitted from the cytoskeleton to ligand-bound integrins acts as an allosteric stabilizer of the extended-open, high-affinity state3. Since cytoskeletal forces in migrating cells are generated by centripetal “retrograde flow” of F-actin from the cell leading edge, where integrin-based adhesions are initiated4,5, this model predicts that F-actin flow should align and orient activated, ligand-bound integrins in integrin-based adhesions. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrin chimeras in migrating fibroblasts shows that integrins are aligned with respect to the axis of FAs and the direction of F-actin flow, and this alignment requires binding immobilized ligand and talin-mediated linkage to a flowing cytoskeleton. Polarization imaging and Rosetta modelling of chimeras engineered to orient GFP differentially with respect to the integrin headpiece suggest that ligand-bound αVβ3 integrin may be markedly tilted by the force of F-actin flow. These results show that actin cytoskeletal forces actively sculpt an anisotropic molecular scaffold in FAs that may underlie the ability of cells to sense directional ECM and physical cues.