Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T cell function in immunosuppressive tumors are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumor microenvironment. We devised an in vivo pooled shRNA screen in which shRNAs targeting negative regulators became highly enriched in tumors by releasing a block on T cell proliferation upon tumor antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumor or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family: In tumors, Ppp2r2d knockdown inhibited T cell apoptosis and enhanced T cell proliferation as well as cytokine production. Key regulators of immune function can thus be discovered in relevant tissue microenvironments.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Advances in microfabrication and nanofabrication are opening new opportunities to investigate complicated questions of cell biology in ways not before possible. In particular, the spatial regulation of cellular processes can be examined by engineering the chemical and physical environment to which the cell responds. Lithographic methods and selective chemical modification schemes can provide biocompatible surfaces that control cellular interactions on the micron and submicron scales on which cells are organized. Combined with fluorescence microscopy and other approaches of cell biology, a widely expanded toolbox is becoming available. This review illustrates the potential of these integrated engineering tools, with an emphasis on patterned surfaces, for investigating fundamental mechanisms of receptor-mediated signaling in cells. We highlight progress made with immune cells and in particular with the IgE receptor system, which has been valuable for developing technology to gain new information about spatial regulation in signaling events.

Plasma membranes are highly dynamic structures, with key molecular interactions underlying their functionality occurring at nanometre scales. A fundamental challenge in biology is to observe these interactions in living cells. Although fluorescence microscopy has enabled advances in characterizing molecular distributions in cells, optical techniques are restricted by the diffraction limit. We address this limitation with an approach based on zero-mode waveguides (ZMWs), which are optical nanostructures that confine fluorescence excitation to sub-diffraction volumes. Successful use of ZMWs with cell membranes is reported in this paper. We demonstrate that plasma membranes from live cells penetrate these nanostructures. Cellular exploration of the nanoapertures depends heavily on actin filaments but not on microtubules. Thus, membranes enter the confined excitation volume, and diffusion of individual fluorescent lipids can be monitored. Through fluorescence correlation spectroscopy, we compared DiIC12 and DiIC16 fluorescent labels incorporated into plasma membranes and found distinctive diffusion behaviours. These results show that the use of optical nanostructures enables the measurement of membrane events with single molecule resolution in sub-diffraction volumes.

Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc RI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc RI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc RI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc RI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (␣5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc RI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc RI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc RI ␤ subunit but enhances stimulated Ca 2؉ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc RI has a net positive effect on Fc RI signaling.FceRI ͉ Lyn ͉ nanobiotechnology ͉ paxillin ͉ supported lipid bilayers C ells have evolved to respond to their chemical and physical environment. Chemical stimulants in the form of soluble or surface-bound ligands are recognized by specific cell surface receptors, and physical cues are sensed by integrins that bind to extracellular matrix proteins on surrounding substrates (1). Cross-talk between intracellular signaling pathways that are initiated by integrins and by ligand receptors has been clearly demonstrated, although spatial aspects of these processes have not been defined. Surfaces patterned on the micrometer scale offer the opportunity to separate regions that bind integrins from those that present ligands to cell surface receptors and thereby delineate respective cytoskeletal connections.The subject of our study is the IgE receptor (Fc RI), which is a member of the family of multisubunit immune recognition receptors that includes antigen receptors on B cells and T cells. This family of receptors has conserved structural features and similarly initiates intracellular signaling in response to multivalent ligands (antigens) that activate cells by clustering cell surface receptors. Fc RI receptors are found primarily in mast cells and basophi...

Ca2+ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca2+ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca2+ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca2+ oscillations throughout the cell that usually follow the initiating Ca2+ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca2+ puffs at the region of cell contact. Initiating Ca2+ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca2+ release from endoplasmic reticulum stores and to extracellular Ca2+, but they do not depend on store-operated Ca2+ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response.

Supported lipid bilayers are an important biomolecular tool for characterizing immunological synapses. Immobilized bilayers presenting tethered ligands on planar substrates have yielded both spatio-temporal and structural insights into how T cell receptors (TCRs) reorganize during the initial formation of synapses upon recognition of peptide antigen bound to major histocompatibility complex (MHC) molecules. The prototypical configuration of these assays, however, limits the extent to which the kinetics and structure of the supramolecular activation clusters of the synapse (that occur in seconds or minutes) can be related to subsequent complex cellular responses, such as cytokine secretion and proliferation, occurring over hours to days. Here we describe a new method that allows correlative measures of both attributes with single-cell resolution by using immobilized lipid bilayers and tethered ligands on the surface of dense arrays of subnanoliter wells. This modification allows each nanowell to function as an artificial antigen-presenting cell (APC), and the synapses formed upon contact can be imaged by fluorescence microscopy. We show that the lipid bilayers remain stable and mobile on the surface of the PDMS, and that modifying the ligands tethered to the bilayer alters the structure of the resulting synapses in expected ways. Finally, we demonstrate that this approach allows the subsequent characterization of secreted cytokines from the activated human T cell clones by microengraving in both antigen- and pan-specific manners. This new technique should allow detailed investigations on how biophysical and structural aspects of the synapse influence the activation of individual T cells and their subsequent complex functional responses.

Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.