Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR).

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn(2+), and that Zn(2+) is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn(2+) chelation and bound Zn(2+) with high selectivity against Ca(2+) and Mg(2+). When added to cultured ß cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn(2+) concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled ß cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn(2+)/insulin release occurred largely in small groups of adjacent ß cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca(2+) elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca(2+) signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn(2+)-containing granules, ZIMIR may find applications in ß-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

A novel sensor kinase is required for Bordetella bronchiseptica to colonize the lower respiratory tract.

Bacterial virulence is influenced by the activity of two-component regulator systems (TCSs), which consist of membrane-bound sensor kinases that allow bacteria to sense the external environment and cytoplasmic, DNA-binding response regulator proteins that control appropriate gene expression. Respiratory pathogens of the Bordetella genus require the well-studied TCS BvgAS to control the expression of many genes required for colonization of the mammalian respiratory tract. Here we describe the identification of a novel gene in Bordetella bronchiseptica, plrS, the product of which shares sequence homology to several NtrY-family sensor kinases and is required for B. bronchiseptica to colonize and persist in the lower, but not upper, respiratory tract in rats and mice. The plrS gene is located immediately 5' to and presumably cotranscribed with a gene encoding a putative response regulator, supporting the idea that PlrS and the product of the downstream gene may compose a TCS. Consistent with this hypothesis, the PlrS-dependent colonization phenotype requires a conserved histidine that serves as the site of autophosphorylation in other sensor kinases, and in strains lacking plrS, the production and/or cellular localization of several immune-recognized proteins is altered in comparison to that in the wild-type strain. Because plrS is required for colonization and persistence only in the lower respiratory tract, a site where innate and adaptive immune mechanisms actively target infectious agents, we hypothesize that its role may be to allow Bordetella to resist the host immune response.