Transient protein accumulation at the center of the T cell antigen-presenting cell interface drives efficient IL-2 secretion.

Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center of the interface of T cells activated by antigen-presenting cells. We have determined that it is composed of multiple complexes of a supramolecular volume of up to 0.5 µm3 and associated with extensive membrane undulations. To determine cSMAC function, we have systematically manipulated the localization of three adaptor proteins, LAT, SLP-76, and Grb2. cSMAC localization varied between the adaptors and was diminished upon blockade of the costimulatory receptor CD28 and deficiency of the signal amplifying kinase Itk. Reconstitution of cSMAC localization restored IL-2 secretion which is a key T cell effector function as dependent on reconstitution dynamics. Our data suggest that the cSMAC enhances early signaling by facilitating signaling interactions and attenuates signaling thereafter through sequestration of a more limited set of signaling intermediates.

Cells receive dozens of signals at different times and in different places. Integrating incoming information and deciding how to respond is no easy task. Signaling molecules on the cell surface pass messages inwards using chemical messengers that interact in complicated networks within the cell. One way to unravel the complexity of these networks is to look at specific groups of signaling molecules in test tubes to see how they interact. But the interior of a living cell is a very different environment. Molecules inside cells are tightly packed and, under certain conditions, they interact with each other by the thousands. They form structures known as 'supramolecular complexes', which changes their behavior. One such supramolecular complex is the 'central supramolecular activation cluster', or cSMAC for short. It forms under the surface of immune cells called T cells when they are getting ready to fight an infection. Under the microscope, the cSMAC looks like the bullseye of a dartboard, forming a crowd of signaling molecules at the center of the interface between the T cell and another cell. Its exact role is not clear, but evidence suggests it helps to start and stop the signals that switch T cells on. The cSMAC contains two key protein adaptors called LAT and SLP-76 that help to hold the structure together. So, to find out what the cSMAC does, Clark et al. genetically modified these adaptors to gain control over when the cSMAC forms. Clark et al. examined mouse T cells using super-resolution microscopy and electron microscopy, watching as other immune cells delivered the signal to switch on. As the T cells started to activate, the composition of the cSMAC changed. In the first two minutes after the cells started activating, the cSMAC included a large number of different components. This made T cell activation more efficient, possibly because the supramolecular complex was helping the network of signals to interact. Later, the cSMAC started to lose many of these components. Separating components may have helped to stop the activation signals. Understanding how T cells activate could lead to the possibility of turning them on or off in immune-related diseases. But these findings are not just relevant to immune cells. Other cells also use supramolecular complexes to control their signaling. Investigating how these complexes change over time could help us to understand how other cell types make decisions.

New milbemycin metabolites from the genetically engineered strain Streptomyces bingchenggensis BCJ60.

Two new milbemycin derivatives, 27-methoxylmilbemycin α31 (1) and 27-oxomilbemycin α31 (2), were isolated from the genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were determined by 1D NMR, 2D NMR and HR-ESI-MS spectral analysis, and comparison with previous reports. The acaricidal and nematocidal capacities of compounds 1 and 2 were evaluated against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, respectively. The results showed that the two new macrocyclic lactones 1 and 2 possessed potent acaricidal and nematocidal activities.

Development and optimization of a process for automated recovery of single cells identified by microengraving.

Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost-effective manner, but connecting these systems to the existing platforms used in routine high-throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody-producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high-throughput, single-cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099-01) were sorted with 100% accuracy (50 clones of each) in approximately 2 h, and the cells retained viability.