Travis I. Moore

Robust Spatial Sensing of Mating Pheromone Gradients by Yeast Cells

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.

PhotoPoint photodynamic therapy promotes stabilization of atherosclerotic plaques and inhibits plaque progression.

OBJECTIVES The purpose of this study was to determine how photodynamic therapy (PDT) promotes stabilization and reduction of regional atherosclerosis. BACKGROUND Photodynamic therapy, a combination of photosensitizer and targeted light to promote cell apoptosis, has been shown to reduce atherosclerotic plaque inflammation. METHODS Forty New Zealand White rabbits were fed with cholesterol. The iliac arteries were balloon denuded and randomized to receive either PhotoPoint PDT treatment (photosensitizer and light) (Miravant Medical Technologies, Santa Barbara, California), photosensitizer (MV0611) alone, or light alone and were then compared at 7 and 28 days. Arteries were examined for evidence of plaque volume, cell number, macrophage and smooth muscle cell (SMC) content, and plaque cell proliferation. RESULTS Compared with contralateral iliac artery controls at 7 days, plaque progression was reduced by approximately 35% (p < 0.01); plaque progression was further reduced to approximately 53% (p < 0.01) by 28 days, leading to an increase in lumen patency (p < 0.05). At 7 days after PDT, percent plaque area occupied by macrophages decreased by approximately 98% (p < 0.001) and SMCs by approximately 72% (p < 0.05). At 28 days after PDT, removal of macrophages was sustained (approximately 92% decrease, p < 0.001) and plaques were repopulated with non-proliferating SMCs (approximately 220% increase, p < 0.001). There was no evidence of negative or expansive arterial remodeling, thrombosis, or aneurysm formation. CONCLUSIONS 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.

Frontline: The p85α isoform of phosphoinositide 3-kinase is essential for a subset of B cell receptor-initiated signaling responses

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 p85α regulatory isoform of PI3K or in wild‐type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85α to early signaling events has not been fully investigated. Here we show that B cells lacking p85α have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85α results in partial reductions in Ca2+ mobilization and IκB 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 p85α‐associated catalytic subunits. Notably, addition of phorbol ester restores BCR‐mediated proliferation in p85α‐deficient cells but not wild‐type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85α is a failure to activate diacylglycerol‐regulated signaling enzymes, most likely protein kinase C.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

Significance Integrins are adhesion receptors linking cells to their environment, which function as sensors of physical and chemical information to regulate development, immune response, and vascular function. How integrins receive and transduce directional forces including flow or tissue tension has remained elusive. We used polarization-based microscopy techniques to discover that activated αVβ3 integrins are aligned with one another in focal adhesions in migrating fibroblasts. Integrin coalignment is sensitive to mechanical resistance of its ligand and coupling to a dynamic F-actin cytoskeleton, consistent with the “cytoskeleton force model” for integrin activation. Our work suggests that activated integrins are actively ordered at the molecular scale by cellular forces, which may underlie their ability to sense directional forces in their environment to mediate critical functions. 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 contractility-driven “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.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

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.Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here the authors show that actin flow can orient cell surface integrins during leukocyte migration, suggesting integrin activation by cytoskeletal force.

Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction

G-proteins, heterotrimeric and Cdc42, modulate in a ligand-dependent fashion two fundamental cell polarity behaviors (projection bending growth and second projection formation) in response to gradient directional change.

Signaling Regulated Endocytosis and Exocytosis Lead to Mating Pheromone Concentration Dependent Morphologies in Yeast

Polarized cell morphogenesis requires actin cytoskeleton rearrangement for polarized transport of proteins, organelles and secretory vesicles, which fundamentally underlies cell differentiation and behavior. During yeast mating, Saccharomyces cerevisiae responds to extracellular pheromone gradients by extending polarized projections, which are likely maintained through vesicle transport to (exocytosis) and from (endocytosis) the membrane. We experimentally demonstrate that the projection morphology is pheromone concentration‐dependent, and propose the underlying mechanism through mathematical modeling. The inclusion of membrane flux and dynamically evolving cell boundary into our yeast mating signaling model shows good agreement with experimental measurements, and provides a plausible explanation for pheromone‐induced cell morphology.

Application of a new wall-less plate technology to complex multistep cell-based investigations using suspension cells

Despite significant progresses, cell-based assays still have major limitations part to because of their plate format. Here, we present a wall-less plate technology based on unique liquid dynamics named DropArray that takes advantage of hydrophobic and hydrophilic surface properties. Liquid velocities within the DropArray plate were quantified through fluid dynamics simulation and complete retention of suspension cells experimentally demonstrated within the range of simulated shear stresses. Subsequently, we compared the DropArray technology with conventional microtiter plates in a cell-based protein-binding assay. Although the wall-less plate produced similar results with adherent cells, the advantage of the DropArray technology was absolutely clear when semiadherent or suspension cells were used in this multistep experimental procedure. The technology also was evaluated for the cell viability assay and generated similar results to conventional plate format while enabling significant reduction in toxic reagent use. Finally, we developed a DropArray cell-based assay to evaluate a bispecific antibody designed to engage cytotoxic T cells and trigger tumor cell killing. This assay enables for the first time the visualization and quantification of the specific killing events and represents a very powerful tool to further investigate functional aspects of the cancer immunotherapy.

Measuring Integrin Conformational Change on the Cell Surface with Super-Resolution Microscopy

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.

1064-184 Photodynamic therapy reduces atherosclerotic plaque inflammation, induces plaque stabilization and promotes plaque reduction

Va sc ul ar D is ea se , H yp er te ns io n, a nd P re ve nt io n (13.85 mm2) or ESP 24218 (11.7 mm2) compared to mice receiving only saline injections (41.3 mm2)(p<0.02 for both comparisons). Atherosclerosis in mice expressing the control peptide (30.8 mm2) was not significantly different than in mice receiving only the saline injection (p=ns). Conclusion. These results demonstrate an atheroprotective effect of ESP 24218, a 22 amino acid peptide designed to mimic the activity of ApoA-I, in mice.