During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Summary The nature and site of tumor-antigen presentation to immune T cells by bone-marrow-derived cells within the tumor microenvironment remains unresolved. We generated a fluorescent mouse model of spontaneous immuno-evasive breast cancer and identified a subset of myeloid cells with significant similarity to dendritic cells and macrophages that constitutively ingest tumor-derived proteins and present processed tumor antigens to reactive T cells. Using intravital live-imaging, we determined that infiltrating tumor-specific T cells engage in long-lived interactions with these cells, proximal to the tumor. In vitro, these cells capture cytotoxic T cells in signaling-competent conjugates, but do not support full-activation or sustain cytolysis. The spatiotemporal dynamics revealed here implicate non-productive interactions between T cells and antigen presenting cells on the tumor margin.

The systems that refine actomyosin forces during motility remain poorly understood. Septins assemble on the T cell cortex and are enriched at the mid-zone in filaments. Septin knockdown causes membrane blebbing, excess leading edge protrusions, and lengthening of the trailingedge uropod. The associated loss of rigidity permits motility, but cells become uncoordinated and poorly persistent. This also relieves a previously unrecognized restriction to migration through small pores. Pharmacologically rigidifying cells counteracts this effect, and relieving cytoskeletal rigidity synergizes with septin-depletion. These data suggest that septins tune actomyosin forces during motility, and likely regulate lymphocyte trafficking in confined tissues.

The mammalian target of rapamycin (mTOR) is a central controller of cell growth, and it regulates translation, cell size, cell viability, and cell morphology. mTOR integrates a wide range of extracellular and intracellular signals, including growth factors, nutrients, energy levels, and stress conditions. Rheb, a Ras-related small GTPase, is a key upstream activator of mTOR. In this study, we found that Bnip3, a hypoxia-inducible Bcl-2 homology 3 domain-containing protein, directly binds Rheb and inhibits the mTOR pathway. Bnip3 decreases Rheb GTP levels in a manner depending on the binding to Rheb and the presence of the N-terminal domain. Both knockdown and overexpression experiments show that Bnip3 plays an important role in mTOR inactivation in response to hypoxia. Moreover, Bnip3 inhibits cell growth in vivo by suppressing the mTOR pathway. These observations demonstrate that Bnip3 mediates the inhibition of the mTOR pathway in response to hypoxia.Target of rapamycin (TOR), 2 is an evolutionarily conserved serine/threonine kinase that plays a central role in cell growth (1-3). TOR regulates many processes, including protein translation, ribosome biogenesis, autophagy, and metabolism. TOR functions to integrate a wide range of extracellular and intracellular signals to produce a concerted cellular response, such as to stimulate cell growth. For example, mammalian target of rapamycin (mTOR) is activated by growth factor and nutrient availability. In contrast, mTOR is inhibited by cellular energy starvation and various stress conditions, including osmotic stress and hypoxia. These observations established a fundamental importance of mTOR in signal integration in regulation of cell growth.Recent studies have demonstrated that TOR exists in two functionally distinct protein complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2) (3, 4). The two TOR complexes were initially identified in yeast and subsequently were also characterized in Drosophila and mammalian cells. TORC1 contains mTOR, mLST8, PRAS40, and Raptor, whereas TORC2 contains mTOR, mLST8, Rictor, and Sin1 (5-12). TORC1 is responsible for phosphorylation of Thr 389 of S6K1 (ribosomal protein S6 kinase) and 4EBP1 (eukaryote initiation factor 4E-binding protein), two important regulators in protein synthesis (1). In contrast, TORC2 has different substrates and is responsible for phosphorylation of the hydrophobic sites in both AKT and PKC (9, 13). Interestingly, TORC1 but not TORC2 is inhibited by rapamycin (6,8,9). These observations clearly demonstrate that the two TOR complexes have different physiological functions in vivo.Much progress has been made regarding the mechanisms of TORC1 regulation. Tuberous sclerosis complex (TSC) is a genetic disease characterized by benign hamartomas in various tissues (14). Mutations in either the TSC1 or TSC2 tumor suppressor gene are responsible for TSC development. TORC1 is highly activated in TSC tumors or cells with mutation of either TSC1 or TSC2. Both genetic and biochemical data have demonstrated th...

Introduction:In order for T cells to mount an adaptive immune response and enact cell-mediated immunity, they must first successfully detect rare cognate antigen. This detection is achieved by surfacebound T cell receptors (TCRs), binding to peptide-major histocompatibility complexes (pMHC). With some temporal latency, this binding event induces TCR signaling and T cell effector function. In order for TCR recognition to take place, T cells must efficiently survey surfaces of antigen presenting cells (APCs), which may display mainly non-stimulatory pMHC and only rare cognate antigen, in a process involving close (nanometer-scale) membrane apposition. Additionally, those rare pMHC ligands are distributed nonuniformly on subsets of APCs and only within specific lymph nodes. Thus, T cells must solve a classic search tradeoff between speed and sensitivity: faster movements provide larger overall coverage with costs at the level of sensitivity. Successful search, which results in ligand detection, is ultimately required for effector function and T cell-mediated adaptive immune response. Although surface deformations are indicated in this recognition process, the full understanding of search strategy requires real-time full 3-dimensional analysis that has not been possible using fixed or low-resolution approaches.Rationale: It has long been supposed that small microvilli on T cell surfaces are used as sensory organs to enable the search for pMHC, but their strategy has not been amenable to study. We used time-resolved lattice light sheet (LLS) microscopy and Qdot-enabled synaptic contact mapping (SCM) microscopy to show how microvilli on the surface of T cells search opposing cells and surfaces prior to and during antigen-recognition. Results:In characterizing microvilli movement on T cell surfaces, we uncovered fractal organization of the microvilli, suggesting consistent coverage across scales. We found that their movements surveyed the majority of opposing space within one minute, which is equivalent to the roughly one minute half-life of T cell-APC contacts in vivo. Individual microvilli local dwell times were sufficiently long to permit discrimination of pMHC half-lives. Protrusion density was similar in non-synapse and synapse regions and did not change appreciably during synapse development, suggesting that T cells did not "intensify" search upon recognition. TCR recognition resulted in selective stabilization of receptor-occupied protrusions as seen by longer microvilli dwell times in synapse regions with pMHC and increased persistence of TCR-occupied contacts. Microvillar scanning in synapse regions lacking pMHC showed dynamics similar to non-synaptic regions, supporting the dependence of TCR stabilization on ligand recognition. Subsequent TCR movements took place upon the stabilized protrusions, even while transient ones tested new regions. In the absence of tyrosine kinase signaling, microvillar search and TCR-occupied protrusion stabilization continued. Intrinsic stabilization was also independent of the ac...