Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors.

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development.

Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 µM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 µM with a theoretical detection limit of 3.4 µM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 µM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

The TAO kinase KIN-18 regulates contractility and establishment of polarity in the C. elegans embryo.

Cell polarity is crucial for many aspects of cell and developmental biology. Cytoskeleton remodeling plays an essential role in the establishment of cell polarity. In the Caenorhabditis elegans one-cell embryo, while the actomyosin cytoskeleton is required for asymmetric localization of the PAR proteins, anterior PAR proteins exert a feedback regulation on contractility. Here we identify the TAO kinase KIN-18 as a regulator of cortical contractility in the early embryo. KIN-18 negatively regulates cortical contractions in a RHO-1 dependent manner and regulates RHO-1 cortical localization. KIN-18 contributes to polarity establishment by regulating the position of the boundary between anterior and posterior PAR proteins. Although KIN-18 is involved in polarity establishment, depletion of KIN-18 restores contractions in a par-3 mutant indicating that kin-18 is epistatic to par-3. We suggest a model in which KIN-18 provides a link between the cytoskeleton remodeling and polarity machineries, uncovering a role for TAO kinases in the regulation of cell polarity.

Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97 (ref. 3), a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes. However, the role and relevance of p97 in nucleus formation have remained controversial. Here we show that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation.

Selection of Structure-Switching DNA Aptamers Binding Soluble Small Molecules and SPR Validation of Enrichment.

Capture-SELEX is an effective molecular strategy enabling the discovery of structure-switching aptamers, which might find useful application in molecular detection or separation. We here provide a protocol to perform capture-SELEX for DNA aptamers binding soluble small molecules, which includes a straightforward functional validation by SPR. The SELEX strategy here described is adaptable to any water-soluble molecular target and might foster the development of DNA aptamers binding therapeutic small molecules, at the great advantage of clinical bioanalytics.

Erratum to: Selection of Structure-Switching DNA Aptamers Binding Soluble Small Molecules and SPR Validation of Enrichment.

Erratum to: Chapter 13 in: Giampaolo Zuccheri (ed.), DNA Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1811, https://doi.org/10.1007/978-1-4939-8582-1_13.

More DNA-Aptamers for Small Drugs: A Capture-SELEX Coupled with Surface Plasmon Resonance and High-Throughput Sequencing.

To address limitations in the production of DNA aptamers against small molecules, we introduce a DNA-based capture-SELEX (systematic evolution of ligands by exponential enrichment) protocol with long and continuous randomized library for more flexibility, coupled with in-stream direct-specificity monitoring via SPR and high throughput sequencing (HTS). Applying this capture-SELEX on tobramycin shows that target-specificity arises at cycle number 8, which is confirmed by sequence convergence in HTS analysis. Interestingly, HTS also shows that the most enriched sequences are already visible after only two capture-SELEX cycles. The best aptamers displayed K(D) of approximately 200 nM, similar to RNA and DNA-based aptamers previously selected for tobramycin. The lowest concentration of tobramycin detected on label-free SPR experiments with the selected aptamers is 20-fold smaller than the clinical range limit, demonstrating suitability for small-drug biosensing.

Hybridization chain reaction performed on a metal surface as a means of signal amplification in SPR and electrochemical biosensors.

A more specific and intense signal is desirable for most kinds of biosensors for biomedical or environmental applications, and it is especially so for label-free biosensors. In this paper, we show that hybridization chain reaction (HCR) can be exploited for the easily detectable accumulation of nucleic acids on metal surfaces as an event triggered by specific recognition between a probe and a target nucleic acid. We show that this process could be exploited to increase the sensitivity in the detection of nucleic acids derived from a pathogenic microorganism. This strategy can be straightforwardly implemented on SPR biosensors (commercial or custom-built) or on label-free electrochemical biosensors. Together with signal amplification, HCR can serve as a confirmation of the specificity of target recognition, as it involves the specific matching with a separate base sequence in the target nucleic acid. Furthermore, the kinetics of the target binding and the HCR can be easily distinguished from each other, providing an additional means of confirmation of the specific recognition.