Matthew D. Sekedat

Structure–function mapping of a heptameric module in the nuclear pore complex

Integration of EM, protein–protein interaction, and phenotypic data reveals novel insights into the structure and function of the nuclear pore complex’s ∼600-kD heptameric Nup84 complex.

GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome.

Previous studies have led to a picture wherein the replication of DNA progresses at variable rates over different parts of the budding yeast genome. These prior experiments, focused on production of nascent DNA, have been interpreted to imply that the dynamics of replication fork progression are strongly affected by local chromatin structure/architecture, and by interaction with machineries controlling transcription, repair and epigenetic maintenance. Here, we adopted a complementary approach for assaying replication dynamics using whole genome time‐resolved chromatin immunoprecipitation combined with microarray analysis of the GINS complex, an integral member of the replication fork. Surprisingly, our data show that this complex progresses at highly uniform rates regardless of genomic location, revealing that replication fork dynamics in yeast is simpler and more uniform than previously envisaged. In addition, we show how the synergistic use of experiment and modeling leads to novel biological insights. In particular, a parsimonious model allowed us to accurately simulate fork movement throughout the genome and also revealed a subtle phenomenon, which we interpret as arising from low‐frequency fork arrest.

Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments.

Direct Measurement of Cathepsin B Activity in the Cytosol of Apoptotic Cells by an Activity-Based Probe

Cells control their own death through a program termed apoptosis, which is indispensable for development and homeostasis in all metazoans. Lysosomal cysteine proteases are not normally thought of as participating in apoptosis; however, recent reports have shown that the cathepsin proteases can be released from the lysosome during apoptosis, where they can participate in cell death. We report here the development of an activity-based probe that, under optimized conditions, reports on cathepsin B activity only in apoptotic cells by reading out the release of cathepsin B from the lysosomes. Biochemical characterization of apoptosis in cells from cathepsin B null mice shows delayed and suboptimal activation of caspases. Our data further supports a role for cathepsin B in the cytosol as a positive regulator of a cell death feed-forward loop and provides a chemical tool for future investigations.