Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

Hidden Host Mortality from an Introduced Parasitoid: Conventional and Molecular Evaluation of Non-Target Risk

Hidden trophic interactions are important in understanding food web ecology and evaluating the ecological risks and benefits associated with the introduction of exotic natural enemies in classical biological control programs. Although non-target risk is typically evaluated based on evidence of successful parasitism, parasitoid-induced host mortality not resulting in visible evidence of parasitism (i.e., nonreproductive effects) is often overlooked. The adventive establishment of Trissolcus japonicus, an exotic parasitoid of the introduced stink bug Halyomorpha halys, provides an opportunity to investigate the total impact of this parasitoid on target and non-target hosts in the field. We developed a new methodology to measure nonreproductive effects in this system, involving a species-specific diagnostic PCR assay for T. japonicus. We applied this methodology to field-deployed eggs of four pentatomid species, coupled with traditional rearing techniques. Nonreproductive effects were responsible for the mortality of an additional 5.6% of H. halys eggs due to T. japonicus, and were even more substantial in some of the non-target species (5.4–43.2%). The observed hidden mortality of native non-target species from an introduced parasitoid could change predictions about direct and indirect ecological interactions and the efficacy of biological control of the target pest