Lipoteichoic acid (LTA) is one of two anionic polymers on the surface of the gram-positive bacterium Staphylococcus aureus. LTA is critical for the bacterium-host cell interaction and has recently been shown to be required for cell growth and division. To determine additional biological roles of LTA, we found it necessary to identify permissive conditions for the growth of an LTA-deficient mutant. We found that an LTA-deficient S. aureus ⌬ltaS mutant could grow at 30°C but not at 37°C. Even at the permissive temperature, ⌬ltaS mutant cells had aberrant cell division and separation, decreased autolysis, and reduced levels of peptidoglycan hydrolases. Upshift of ⌬ltaS mutant cells to a nonpermissive temperature caused an inability to exclude Sytox green dye. A high-osmolarity growth medium remarkably rescued the colony-forming ability of the ⌬ltaS mutant at 37°C, indicating that LTA synthesis is required for growth under low-osmolarity conditions. In addition, the ⌬ltaS mutation was found to be synthetically lethal with the ⌬tagO mutation, which disrupts the synthesis of the other anionic polymer, wall teichoic acid (WTA), at 30°C, suggesting that LTA and WTA compensate for one another in an essential function.

HighlightsPazopanib, in addition to dasatinib and statins, activates the Hippo pathway.Pazopanib induces the proteasomal degradation of YAP/TAZ.YAP/TAZ inhibitors reduce viability of YAP/TAZ-dependent breast cancer cells.YAP/TAZ inhibitors sensitize cancer cells to anti-cancer drugs.

Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore–spindle binding and chromosome segregation are mediated by the multi-copy KNL1Spc105, MIS12Mtw1 and NDC80Ndc80 complexes that form the so-called KMN network. KMN–spindle attachment is regulated by the Aurora BIpl1 and MPS1Mps1 kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.

SummaryProper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment) [1]. The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust [2–4] and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation [1]. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface [1, 5], and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex [6], in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end [3, 7]. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.

Lysylphosphatidylglycerol (LPG) is a basic phospholipid in which L-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The K m values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 mM and 6?9 mM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.

No abstract

Genetic screening for suppressor mutants has been successfully used to identify important signaling regulators. Using an analogy to genetic suppressor screening, we developed a chemical suppressor screening method to identify inhibitors of the Wnt/β-catenin signaling pathway. We used zebrafish embryos in which chemically induced β-catenin accumulation led to an "eyeless" phenotype and conducted a pilot screening for compounds that restored eye development. This approach allowed us to identify geranylgeranyltransferase inhibitor 286 (GGTI-286), a geranylgeranyltransferase (GGTase) inhibitor. Our follow-up studies showed that GGTI-286 reduces nuclear localization of β-catenin and transcription dependent on β-catenin/T cell factor in mammalian cells. In addition to pharmacological inhibition, GGTase gene knockdown also attenuates the nuclear function of β-catenin. Overall, we validate our chemical suppressor screening as a method for identifying Wnt/β-catenin pathway inhibitors and implicate GGTase as a potential therapeutic target for Wnt-activated cancers.

A series of A-ring-modified lamellarin N analogues were designed, synthesized, and evaluated as potential noncovalent inhibitors of the EGFR T790M/L858R mutant, a causal factor in the drug-resistant non-small cell lung cancer. Several water-soluble ammonium- or guanidinium-tethered analogues exhibited good kinase inhibitory activities. The most promising analogue, 14f, displayed an excellent inhibitory profile against the T790M/L858R mutant [IC (WT) = 31.8 nM; IC (T790M/L858R) = 8.9 nM]. The effects of A-ring-substituents on activity were rationalized by docking studies.