Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium.

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells-the progeny of basal stem cells-are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. Here, we investigate how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. We find they gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity.

Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss.

Organ renewal is governed by the dynamics of cell division, differentiation and loss. To study these dynamics in real time, we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem-cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hr and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. By applying a pipeline for movie processing and analysis, we uncover new and intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of adult organ renewal.

Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase.

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.