[
International Worm Meeting,
2015]
A series of events during the meiotic cell cycle enables the segregation of homologous chromosomes to form haploid gametes. Early in meiosis, chromosomes are reorganized around a central axis, and then pair, synapse, and recombine with their homologs. In C. elegans, a meiosis-specific paralog of the DNA damage checkpoint kinase CHK-2 is required for pairing, synapsis, and recombination. We have found that CHK-2 promotes pairing and synapsis by phosphorylating a family of zinc finger proteins (HIM-8, ZIM-1, ZIM-2 and ZIM-3) that bind to pairing centers, specialized regions of each chromosome that play essential roles in meiotic chromosome dynamics. Phosphorylation of these proteins by CHK-2 primes their recruitment of the Polo-like kinase PLK-2 to promote pairing and synapsis. A phospho-specific antibody has revealed that CHK-2 activity normally declines once all chromosomes have accomplished synapsis and crossing-over, but is prolonged in mutants that disrupt crossover formation, suggesting that CHK-2 activity is regulated by a feedback circuit that monitors crossover formation. Interestingly, CHK-2 activity is not extended in mutants that disrupt the meiotic HORMA domain proteins HIM-3, HTP-1, or HTP-3, which are components of meiotic chromosome axes, despite their inability to form crossovers. Deleting HIM-3 or HTP-1/2 suppresses the extension of CHK-2 activity in other meiotic mutants, indicating that each of these proteins plays an essential role in sensing and/or signaling meiotic defects for CHK-2 regulation. Feedback control is also abrogated by mutations in HTP-3 that prevent recruitment of HTP-1/2 or HIM-3 to the chromosome axis, indicating that interactions among these axis-associated proteins are essential for meiotic feedback regulation. Our findings establish the molecular basis for checkpoint mechanisms targeting CHK-2 that coordinate meiotic chromosome dynamics with cell cycle progression. .
[
Antimicrob Agents Chemother,
2010]
Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 microM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.
Kostow, Nora, Rog, Ofer, Kim, Yumi, Corbett, Kevin D., Dernburg, Abby F., Rosenberg, Scott C.
[
International Worm Meeting,
2015]
Segregation of chromosomes in meiosis requires their dramatic, stepwise reorganization during meiotic prophase. Each chromosome must pair, synapse, and recombine with its homolog to achieve the stable bivalent structures that biorient and then divide during Meiosis I. A fundamental mystery is how these events are coordinated during the meiotic cell cycle. Pairing, synapsis, and recombination depend on the formation of linear "axes" along each chromosome at meiotic entry, followed by assembly of the synaptomemal complex (SC) between paired axes. Chromosome axes in C. elegans are comprised of cohesins and four related HORMA domain proteins: HIM-3, HTP-1, HTP-2, and HTP-3. We recently reported that the largest of these proteins, HTP-3, recruits the other three paralogs through short peptide sequences (closure motifs) in its C-terminal tail, which are bound by the HORMA domains of HTP-1, HTP-2, and HIM-3 (Kim, Rosenberg, et al., 2014). Here we show that these interactions are dynamically regulated by phosphorylation of the closure motifs in HTP-3 by two meiotic kinases, CHK-2 and PLK-2. Phosphorylation of the four central motifs reduces their binding affinity for HIM-3, and occurs in two temporally distinct waves. In early meiosis, phosphorylation along the entire axis by CHK-2 is required for efficient synapsis. A second wave of phosophorylation by PLK-2 occurs after crossover formation, and specifies the "short arm" of the bivalent where cohesion will be released during the first meiotic division. This second wave of HTP-3 phosphorylation requires both crossover formation and recruitment of PLK-2 to the chromosomes through a binding site in a SC component, SYP-1. Thus, phosphorylation-dependent regulation of HORMA domain protein assembly promotes dynamic remodeling of chromosome axes during meiotic progression, and is essential for proper segregation of holocentric chromosomes in C. elegans meiosis.