Highlights d Time-dependent stem cell depletion levels off in the old brain via increased quiescence d Age minimally changes the neural stem cell transcriptome d Once-activated neural stem cells perform similar in the old and young brain d The old niche keeps stem cells quiescent via inflammation and Wnt activity regulation

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We examined responses of the B-cell antigen receptor-dependent intracellular signaling network to targeted perturbations induced through siRNA-mediated depletion of select signaling intermediates. The constituent nodes displayed graded sensitivities, which resulted from the differential effects of perturbations on the kinetic and quantitative aspects of phosphorylation at each node. By taking the rate of initial phosphorylation, rate of subsequent dephosphorylation, and the total intensity of phosphorylation at each node as separate signaling parameters, we generated data-driven models that accurately predicted the cellular responses of apoptosis, proliferation, and cytokine secretion. Importantly, the effects of perturbation on the primary target alone did not yield successful models. Rather, it also required incorporation of secondary effects on many other nodes. A significant feature of these models was that the three signaling parameters derived from each node functioned largely as independent entities, making distinctive contributions to the cellular response. Thus, the kinetic and quantitative features of phosphorylation at a node appear to play discrete roles during signal processing.

Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.