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Gehring, K.

Publications and source records attributed to Gehring, K..

11 recordsLinked to original sources

Bumblebees flexibly adjust learning and decision strategies to sensory information content in a foraging task

When making decisions, humans and animals often rely on multiple sensory cues simultaneously. These provide complementary sources of information, which can help overcome ambiguity and noise, and increase the accuracy of decisions. While most studies have focused on the benefits of multimodal and within-mode integration for learning and decision making, their costs have received less attention. Processing, learning and memorizing multiple inputs requires more neural resources than for single cues, and might also require more time. In this study, we tested whether insects trade off the costs and benefits of learning multiple cues in a foraging task, using the buff-tailed bumblebee Bombus terrestris. To maximize comparability between cues, we presented combinations of visual-only features, as found on natural flowers: colours of varying discriminability, paired with shapes or patterns. We found that the bees relied exclusively on colours when these were easy to discriminate, and did not learn pattern or shape features presented simultaneously. With harder to discriminate colours, the bees learned both colour and shape or pattern features. Our results demonstrate that bumblebees flexibly adjust their learning strategies when presented with visual features of varying discriminability, to balance the costs and benefits of multi-cue learning. Our analysis of the learning rates of the bees with multi- and single attribute stimuli suggests that blocking could serve as a mechanism to implement this strategy switch. These results shed light on trade-offs in learning and decision making with multiple cues, and can directly be compared to studies in other insects, animals and humans. Significance StatementWhen making decisions, animals often rely on multiple sensory cues simultaneously. These complementary sources of information can increase decision accuracy. Unlike the benefits of cue integration, their costs have received less attention. In this study, we tested whether insects trade off the costs and benefits of learning multiple cues in a foraging task. We trained buff-tailed bumblebees, Bombus terrestris, to feature combinations of natural flowers. Bees flexibly switched from linear integration to winner-takes-all decision strategies depending on the sensory information available, thereby balancing accuracy and time investment in the task. We find that this switch can be explained by the cognitive phenomenon of blocking; providing the ground for future investigations into the underlying neural mechanisms of flexible decision strategies.

animal behavior and cognition↗

Small heat shock proteins (sHSPs) identified in nodules of tropical woody legumes.

Biological nitrogen fixation (BNF) is a primary input of nitrogen to natural and agricultural systems globally. BNF is a temperature-dependant enzymatic process and can be conducted by microbes (including Rhizobia) hosted symbiotically in root nodules of some plants. Heat shock proteins (Hsps) have been implicated in the process of acquiring thermotolerance or acclimating to elevated temperature, as they play a vital role in maintaining cell integrity and homeostasis during heat stress. Although the BNF response to temperature may crucially impact future ecosystem productivity in the face of global climate change, little is known about Hsp expression in nodules of N-fixing non-agricultural species, such as tropical N-fixing trees in the Fabaceae family. This project aimed to characterize small (15-20 kDa) Hsp (sHsp) expression in nodule tissue to examine the biochemical mechanisms of heat response in these tissues. To first identify Hsps in nodule tissues, Vachellia farnesiana and Acacia confusa nodules were excised, heat shock was induced, and protein content was isolated via chemical treatment before separation of protein species and analysis with SDS-PAGE. Two polyacrylamide gels yielded bands in the 15-20 kDa region that displayed differential Coomassie staining, which were sent for further characterization by HPLC-MS analysis for protein sequencing. Ten rhizobial sHsps were detected in these samples in addition to seven Acacia sHsps when compared independently to reference rhizobial and plant proteome databases. In an attempt quantify relative expression of Hsps in nodule and root tissue, we performed western blot experiments using Anti-Hsp20 antibodies raised against human and mouse Hsp proteins, with anti-beta actin loading control. While nonuniform beta-actin expression across tissue types (A. confusa nodules versus root control) prevented quantitative analysis, the experiments validated that Hsp20s are expressed in Acacia nodules as well as in root tissue. These experiments provide a foundation for future studies to determine variation in responses to key stressors predicted to increase with global climate change and help determine the implications of warming across the tropics and beyond. Proteomics data are available via ProteomeXchange with identifier PXD055599.

plant biology↗

Understanding the Pathogenicity of Parkin Catalytic Domain Mutants

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinsons disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally characterized and their structural basis revealed, several pathogenic PD mutations found in the catalytic RING2 domain remain poorly understood. Here, we characterize two pathogenic RING2 mutants, T415N and P437L and shed light on the underlying structural causes. For this purpose, we use biochemical in vitro assays in combination with AlphaFold modeling. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. After determining the parkin minimal ubiquitin binding region, we show that both mutants display impaired binding to the ubiquitin molecule charged onto the E2 enzyme. Finally, we employ the most recent version of AlphaFold 3 to generate a structural model of the phospho-parkin/phospho-ubiquitin/ubiquitin-charged E2 complex. This model consolidates our findings and provides a structural understanding for the pathogenicity of these two parkin variants. A better understanding of the different PD mutations at the molecular level can pave the way for personalized treatments and the design of small molecule therapeutics for the treatment of PD.

biochemistry↗

Enhanced binding of guanylated poly(A) RNA by the LaM domain of LARP1

La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di- nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3,3-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation.

biochemistry↗

Structural insights into regulation of TRPM7 divalent cation uptake by the small GTPase ARL15

Cystathionine-{beta}-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They promote efflux of Mg2+ ions on their own or uptake of divalent cations when coupled to the transient receptor potential ion channel subfamily M member 7 (TRPM7). Recently, ADP-ribosylation factor-like GTPase 15 (ARL15) has been identified as CNNM binding partner and an inhibitor of divalent cation influx by TRPM7. Here, we characterize ARL15 as a GTP-binding protein and demonstrate that it binds the CNNM CBS-pair domain with low micromolar affinity. The crystal structure of the complex between ARL15 GTPase domain and CNNM2 CBS-pair domain reveals the molecular determinants of the interaction and allowed the identification of mutations in ARL15 and CNNM2 mutations that abrogate binding. Loss of CNNM binding prevented ARL15 suppression of TRPM7 channel activity in support of previous reports that the proteins function as a ternary complex. Binding experiments with phosphatase of regenerating liver 2 (PRL2 or PTP4A2) revealed that ARL15 and PRLs compete for binding CNNM, suggesting antagonistic regulation of divalent cation transport by the two proteins.

biochemistry↗

Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

Mutations in Parkin and PINK1 cause an early-onset familial Parkinsons disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.

biochemistry↗

Structural basis for feedforward control in the PINK1/parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinsons disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

biochemistry↗

Crystal structure of a bacterial CNNM magnesium transporter

CBS-pair domain divalent metal cation transport mediators (CNNMs) are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. CNNM proteins are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determined the crystal structure of an archaeal CNNM protein with Mg2+-ATP bound. The structure reveals a novel transmembrane fold for the DUF21 domain, the largest family of domains of unknown function. The protein has a negatively charged cavity that penetrates halfway through the membrane suggesting it functions as a cation transporter. The cytosolic portion of the protein is comprised of highly charged four-helix bundle and a CBS-pair domain. HDX-MS experiments, molecular dynamics, and additional crystal structures show that the cytosolic domains undergo large conformational changes upon nucleotide binding suggesting a mechanism of regulation shared between human and bacterial orthologs. The molecular characterization of CNNM proteins has profound implications for understanding their biological functions in human diseases, including cancer, and in animals, bacteria and plants.

biophysics↗

Poly(A) binding protein is required for mRNP remodeling to form P-bodies in mammalian cells

Compartmentalization of mRNA through formation of RNA granules is involved in many cellular processes, yet it is not well understood. mRNP complexes undergo dramatic changes in protein compositions, reflected by markers of P-bodies and stress granules. Here, we show that PABPC1, albeit absent in P-bodies, plays important role in P-body formation. Depletion of PABPC1 decreases P-body population in unstressed cells. Upon stress in PABPC1 depleted cells, individual P-bodies fail to form and instead P-body proteins assemble on PABPC1-containing stress granules. We hypothesize that mRNP recruit proteins via PABPC1 to assemble P-bodies, before PABPC1 is displaced from mRNP. Further, we demonstrate that GW182 can mediate P-body assembly. These findings help us understand the early stages of mRNP remodeling and P-body formation. Summary statementA novel role of poly(A) binding protein is reported in P-body formation

biochemistry↗

Paip2 associates with PABPC1 on mRNA, and may facilitate PABPC1 dissociation from mRNA upon deadenylation

Poly(A) binding protein cytoplasmic 1 (PABPC1) is an essential translational initiation factor. PABPC1 recognizes proteins through conserved PABPC1-interacting motifs 1 and 2 (PAM1 and PAM2). PABPC1-interacting protein-2 (Paip2) interacts with PABPC1 and modulates its activities. Here, we report that the formation of Paip2/PABPC1 complex protects it from proteasome independent degradation. We also show that PAM2 is critical for Paip2/PABPC1 interaction in vivo, in agreement with the observation that Paip2 requires PAM2 to interact with PABPC1 on mRNA. Lastly, we propose a role for Paip2 in displacing PABPC1 at the final stage of mRNA deadenylation when the poly(A) tail is partly degraded.

biochemistry↗

Loss of PABPC1 is compensated by elevated PABPC4 and correlates with transcriptome changes

Cytoplasmic poly(A) binding protein (PABP) is an essential translation factor that binds to the 3 tail of mRNAs to promote translation and regulate mRNA stability. PABPC1 is the most abundant of several PABP isoforms that exist in mammals. Here, we used the CRISPR/Cas genome editing system to shift the isoform composition in HEK293 cells. Disruption of PABPC1 elevated PABPC4 levels. Transcriptome analysis revealed that the shift in the dominant PABP isoform was correlated with changes in key transcriptional regulators. This study provides insight into understanding the role of PABP isoforms in development and differentiation.

biochemistry↗