Proceedings of the National Academy of Sciences

doi: N/Apmid: N/A

The Asian tiger mosquito Aedes albopictus transmits the dengue virus across the tropics, with a range that has recently expanded across southern Europe and the Americas. Because tiger mosquitoes bite humans during the day, insecticide-treated mosquito nets are of little use in curbing the spread of dengue fever, for which there is no vaccine. Marcus Blagrove et al. (pp. 255–260) attempted to exploit bacteria that naturally infects insects as a means toward replacing wild populations with mosquitoes incapable of spreading dengue. The authors infected A. albopictus with a strain of fruit fly-derived Wolbachia bacteria known as wMel, and found that the bacteria blocked the transmission of dengue virus. Upon mating with infected males, wild female A. albopictus that lack wMel but carrying other strains of Wolbachia produce embryos that rapidly die, a phenomenon called cytoplasmic incompatibility. This incompatibility, the authors reason, could be exploited to replace wild populations with dengue-resistant mosquitoes. In a related report, Xiaoling Pan et al. (pp. 13–14) used molecular analysis to determine how the bacteria enabled virus resistance in the mosquitoes. The authors report that microinjecting a different strain of Wolbachia from A. albopictus into A. aegypti mosquitoes boosted the mosquitoes’ innate immunity. Bacterial infection triggered a spike in oxidative stress, which in turn amped up an immune-related biochemical cascade called the Toll pathway, involved in producing both antioxidants to help counter the stress and antimicrobial peptides …

doi: N/Apmid: 22190492

Binding of EGF to its receptor induces dimerization of the normally monomeric receptor. Activation of its intracellular tyrosine kinase then occurs through the formation of an asymmetric kinase dimer in which one subunit, termed the “receiver” kinase, is activated by interaction with the other subunit, termed the “activator” kinase (Zhang, et al. (2006) Cell 125: 1137–1149). Although there is significant experimental support for this model, the relationship between ligand binding and the mechanics of kinase activation are not known. Here we use luciferase fragment complementation in EGF receptor (EGFR)/ErbB2 heterodimers to probe the mechanics of ErbB kinase activation. Our data support a model in which ligand binding causes the cis-kinase (the EGFR) to adopt the receiver position in the asymmetric dimer and to be activated first. If the EGF receptor is kinase active, this results in the phosphorylation of the trans-kinase (ErbB2). However, if the EGF receptor kinase is kinase dead, the ErbB2 kinase is never activated. Thus, activation of the kinases in the EGFR/ErbB2 asymmetric dimer occurs in a specific sequence and depends on the kinase activity of the EGF receptor.

doi: N/Apmid: 22184213

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. ((2009) Proc Natl Acad Sci USA 106:13124–13129), who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1–4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

doi: N/Apmid: 22184250

The transcription factor zinc-finger protein Miz1 represses TNF-α–induced JNK activation and the repression is relieved upon TNF-α stimulation. However, the underlying mechanism is incompletely understood. Here we report that Miz1 interferes with the ubiquitin conjugating enzyme (E2) Ubc13 for binding to the RING domain of TNF-receptor associated factor 2 (TRAF2), thereby inhibiting the ubiquitin ligase (E3) activity of TRAF2 and suppressing TNF-α–induced JNK activation. Upon TNF-α stimulation, Miz1 rapidly undergoes K48-linked polyubiquitination at Lys388 and Lys472 residues and subsequent proteasomal degradation in a TRAF2-dependent manner. Replacement of Lysine 388 and Lysine 472 by arginines generates a nondegradable Miz1 mutant, which significantly suppresses TNF-α–induced JNK1 activation and inflammation. Thus, our results reveal a molecular mechanism by which the repression of TNF-α–induced JNK activation by Miz1 is de-repressed by its own site-specific ubiquitination and degradation, which may account for the temporal control of TNF-α–JNK signaling.

doi: N/Apmid: 22187460

Toll like receptors (TLRs) use Toll–IL-1 receptor (TIR) domain-containing adapters, such as myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adapter inducing IFN-β (TRIF), to induce activation of transcription factors, including NF-κB, MAP kinases, and IFN regulatory factors. TLR signaling also leads to activation of PI3K, but the molecular mechanism is not understood. Here we have discovered a unique role for B-cell adapter for PI3K (BCAP) in the TLR-signaling pathway. We find that BCAP has a functional N-terminal TIR homology domain and links TLR signaling to activation of PI3K. In addition, BCAP negatively regulates proinflammatory cytokine secretion upon TLR stimulation. In vivo, the absence of BCAP leads to exaggerated recruitment of inflammatory myeloid cells following infections and enhanced susceptibility to dextran sulfate sodium-induced colitis. Our results demonstrate that BCAP is a unique TIR domain-containing TLR signaling adapter crucial for linking TLRs to PI3K activation and regulating the inflammatory response.

doi: N/Apmid: 22184214

G protein-coupled receptors play a central role in signal transduction and were only known to be activated by agonists. Recently it has been shown that membrane potential also affects the activity of G protein-coupled receptors. For the M2 muscarinic receptor, it was further shown that depolarization induces charge movement. A tight correlation was found between the voltage-dependence of the charge movement and the voltage-dependence of the agonist binding. Here we examine whether depolarization-induced charge movement causes a conformational change in the M2 receptor that may be responsible for the voltage-dependence of agonist binding. Using site-directed fluorescence labeling we show a voltage-dependent fluorescence signal, reflecting a conformational change, which correlates with the voltage-dependent charge movement. We further show that selected mutations in the orthosteric site abolish the fluorescence signal and concomitantly, the voltage-dependence of the agonist binding. Surprisingly, mutations in the allosteric site also abolished the voltage-dependence of agonist binding but did not reduce the fluorescence signal. Finally, we show that treatments, which reduced the charge movement or hindered the coupling between the charge movement and the voltage-dependent binding, also reduced the fluorescence signal. Our results demonstrate that depolarization-induced conformational changes in the orthosteric binding site underlie the voltage-dependence of agonist binding. Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltage-dependent agonist binding.

doi: N/Apmid: 22087001

Metazoans and plants use pattern recognition receptors (PRRs) to sense conserved microbial-associated molecular patterns (MAMPs) in the extracellular environment. In plants, the bacterial MAMPs flagellin and elongation factor Tu (EF-Tu) activate distinct, phylogenetically related cell surface pattern recognition receptors of the leucine-rich repeat receptor kinase (LRR-RK) family called FLS2 and EF-Tu receptor, respectively. BAK1 is an LRR-RK coreceptor for both FLS2 and EF-Tu receptor. BAK1 is also a coreceptor for the plant brassinosteroid (BR) receptor, the LRR-RK BRI1. Binding of BR to BRI1 primarily promotes cell elongation. Here, we tune the BR pathway response to establish how plant cells can generate functionally different cellular outputs in response to MAMPs and pathogens. We demonstrate that BR can act antagonistically or synergistically with responses to MAMPs. We further show that the synergistic activities of BRs on MAMP responses require BAK1. Our results highlight the importance of plant steroid homeostasis as a critical step in the establishment of plant immunity. We propose that tradeoffs associated with plasticity in the face of infection are layered atop plant steroid developmental programs.

doi: N/Apmid: 22203980

The authors wish to note the following: “Although our published findings were reproducible in our laboratory and while …

doi: N/Apmid: 22184236

Captive breeding programs are widely used for the conservation and restoration of threatened and endangered species. Nevertheless, captive-born individuals frequently have reduced fitness when reintroduced into the wild. The mechanism for these fitness declines has remained elusive, but hypotheses include environmental effects of captive rearing, inbreeding among close relatives, relaxed natural selection, and unintentional domestication selection (adaptation to captivity). We used a multigenerational pedigree analysis to demonstrate that domestication selection can explain the precipitous decline in fitness observed in hatchery steelhead released into the Hood River in Oregon. After returning from the ocean, wild-born and first-generation hatchery fish were used as broodstock in the hatchery, and their offspring were released into the wild as smolts. First-generation hatchery fish had nearly double the lifetime reproductive success (measured as the number of returning adult offspring) when spawned in captivity compared with wild fish spawned under identical conditions, which is a clear demonstration of adaptation to captivity. We also documented a tradeoff among the wild-born broodstock: Those with the greatest fitness in a captive environment produced offspring that performed the worst in the wild. Specifically, captive-born individuals with five (the median) or more returning siblings (i.e., offspring of successful broodstock) averaged 0.62 returning offspring in the wild, whereas captive-born individuals with less than five siblings averaged 2.05 returning offspring in the wild. These results demonstrate that a single generation in captivity can result in a substantial response to selection on traits that are beneficial in captivity but severely maladaptive in the wild.

doi: N/Apmid: 22190487

Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtubules as demonstrated by in vitro microtubule-binding assay.