RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

The universality of calcium as an intracellular messenger depends on its enormous versatility. Cells have a calcium signalling toolkit with many components that can be mixed and matched to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of calcium being highly toxic. Exceeding its normal spatial and temporal boundaries can result in cell death through both necrosis and apoptosis.

The versatility and universality of calcium signalling

The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor—ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation. A critical event in the initiation of the adaptive

https://www.med.nyu.edu/skirball-lab/dustinlab/pdfs/The%20immunological%20synapse%20%20A%20molecular%20machine%20controlling%20T%20cell%20activation.pdf

The Immunological Synapse: A Molecular Machine Controlling T Cell Activation

The extracellular matrix (ECM) and ECM proteins are important in phenomena as diverse as developmental patterning, stem cell niches, cancer, and genetic diseases. The ECM has many effects beyond providing structural support. ECM proteins typically include multiple, independently folded domains whose sequences and arrangement are highly conserved. Some of these domains bind adhesion receptors such as integrins that mediate cell-matrix adhesion and also transduce signals into cells. However, ECM proteins also bind soluble growth factors and regulate their distribution, activation, and presentation to cells. As organized, solid-phase ligands, ECM proteins can integrate complex, multivalent signals to cells in a spatially patterned and regulated fashion. These properties need to be incorporated into considerations of the functions of the ECM.

/pdf/the-extracellular-matrix-not-just-pretty-fibrils-57y6rgrwav.pdf

The Extracellular Matrix: Not Just Pretty Fibrils

Activation of T cells by antigen-presenting cells (APCs) depends on the complex integration of signals that are delivered by multiple antigen receptors. Most receptor-proximal activation events in T cells were identified using multivalent anti-receptor antibodies, eliminating the need to use the more complex APCs. As the physiological membrane-associated ligands on the APC and the activating antibodies probably trigger the same biochemical pathways, it is unknown why the antibodies, even at saturating concentrations, fail to trigger some of the physiological T-cell responses. Here we study, at the level of the single cell, the responses of T cells to native ligands. We used a digital imaging system and analysed the three-dimensional distribution of receptors and intracellular proteins that cluster at the contacts between T cells and APCs during antigen-specific interactions. Surprisingly, instead of showing uniform oligomerization, these proteins clustered into segregated three-dimensional domains within the cell contacts. The antigen-specific formation of these new, spatially segregated supramolecular activation clusters may generate appropriate physiological responses and may explain the high sensitivity of the T cells to antigen.

Three-dimensional segregation of supramolecular activation clusters in T cells

Every cell contains many families of protein kinases, and may express several structurally related yet genetically distinct kinases of each family. The activity of the serine/threonine protein kinase C (PKC) enzymes has long been implicated in T-cell activation, but it is not known which members of the PKC family regulate the T-cell response to foreign antigens. The activation of T cells by antigen-presenting cells (APCs) is spatially restricted to their site of contact, where receptors on the T cells engage their counter-receptors on the APCs. We used this localized engagement to identify, at the single-cell level, intracellular proteins involved in the activation process. By digital immunofluorescence microscopy, we localized six isoforms of PKC in antigen-specific T-cell clones activated by APCs. Surprisingly, only PKC-theta translocated to the site of cell contact. Accordingly, in vitro kinase activity assays of PKC immunoprecipitates from the conjugates of T cells and APCs showed a selective increase in the activity of PKC-theta, indicating that the translocated enzyme is active. Several modes of partial T-cell activation that failed to cause PKC-theta translocation also failed to cause T-cell proliferation, further suggesting the involvement of PKC-theta in T-cell activation.

Selective modulation of protein kinase C-Θ during T-cell activation

During the productive interaction of T cells with antigen-presenting cells (APCs), engaged receptors, including the T cell antigen receptors and their associated tyrosine kinases, assemble into spatially segregated supramolecular activation clusters (SMACs) at the area of cell contact. Here, we studied intracellular signaling in SMACs by three-dimensional immunofluorescence microscopic localization of CD3, CD45, talin, phosphotyrosine, Lck and phosphorylated ZAP-70 in T cell-APC conjugates. Two distinct phases of spatial-temporal activation, one before and one after SMAC formation, which were separated by a brief state of inactivation caused by CD45, were observed at the T cell-APC contact area. We propose that pre-SMAC signals are sufficient to activate cell adhesion, but not productive T cell responses, which require orchestrated signaling in SMACs.

Staging and resetting T cell activation in SMACs

Antigen (Ag)-specific T helper (Th) cells regulate the proliferation and differentiation of Ag-specific B cells by secreting cytokines and by expressing activating receptors like gp39. In vitro, the cytokines and the activating receptors function in an Ag-nonspecific manner. It is unclear, therefore, how Ag specificity is imposed on B cell responses in physiological Th-B cell interactions. Here we studied, at the single cell level, the interactions between cloned Th cells and small splenic B cells, which served as Ag-specific antigen-presenting cells (APCs) to the Th cells. Digital confocal immunofluorescence microscopy of Th-B cell conjugates revealed significant variability in the molecular and cellular properties of these interactions, in spite of the fact that all the interactions in this system were expected to be Ag specific. After 30 h of incubation B cells began to divide, and this process was entirely dependent on the presence of both Th cells and Ag. Immunofluorescence microscopic studies showed that essentially all the mitotic B cells were bound to Th cells and faced the microtubule organizing center (MTOC) in the Th cells where interleukin 4 was highly concentrated. Other B cells that were bound to the same Th cells but were not close to the Th-MTOC remained in interphase. These results provide the first direct structural and functional evidence that the site of interaction of B cells with Th cells affects their immune response. We propose that, during Ag-induced Th-B cell interactions, B cells that are bound facing the Th-MTOC proliferate preferentially because they are the recipients of locally secreted cytokines. In addition, these B cells may interact with newly expressed receptors, which may also be locally inserted into the Th membrane. The polarized delivery of activating molecules towards the Th-bound APCs may impose functional specificity on effector molecules that otherwise are not Ag specific.

/pdf/small-splenic-b-cells-that-bind-to-antigen-specific-t-helper-120rwt2xta.pdf

Small Splenic B Cells That Bind to Antigen-specific T Helper (Th) Cells and Face the Site of Cytokine Production in the Th Cells Selectively Proliferate: Immunofluorescence Microscopic Studies of Th-B Antigen-presenting Cell Interactions

Hannah Kupfer

Papers

Three-dimensional segregation of supramolecular activation clusters in T cells

Selective modulation of protein kinase C-Θ during T-cell activation

Staging and resetting T cell activation in SMACs

Small Splenic B Cells That Bind to Antigen-specific T Helper (Th) Cells and Face the Site of Cytokine Production in the Th Cells Selectively Proliferate: Immunofluorescence Microscopic Studies of Th-B Antigen-presenting Cell Interactions

Imaging immune cell interactions and functions: SMACs and the Immunological Synapse.