Fluctuations in Intracellular CheY-P Concentration Coordinate Reversals of Flagellar Motors in E. coli

Signal transduction utilizing membrane-spanning receptors and cytoplasmic regulator proteins is a fundamental process for all living organisms, but quantitative studies of the behavior of signaling proteins, such as their diffusion within a cell, are limited. In this study, we show that fluctuations in the concentration of the signaling molecule, phosphorylated CheY, constitute the basis of chemotaxis signaling. To analyze the propagation of the CheY-P signal quantitatively, we measured the coordination of directional switching between flagellar motors on the same cell. We analyzed the time lags of the switching of two motors in both CCW-to-CW and CW-to-CCW switching (∆τCCW-CW and ∆τCW-CCW). In wild-type cells, both time lags increased as a function of the relative distance of two motors from the polar receptor array. The apparent diffusion coefficient estimated for ∆τ values was ~9 µm2/s. The distance-dependency of ∆τCW-CCW disappeared upon loss of polar localization of the CheY-P phosphatase, CheZ. The distance-dependency of the response time for an instantaneously applied serine attractant signal also disappeared with the loss of polar localization of CheZ. These results were modeled by calculating the diffusion of CheY and CheY-P in cells in which phosphorylation and dephosphorylation occur in different subcellular regions. We conclude that diffusion of signaling molecules and their production and destruction through spontaneous activity of the receptor array generates fluctuations in CheY-P concentration over timescales of several hundred milliseconds. Signal fluctuation coordinates rotation among flagella and regulates steady-state run-and-tumble swimming of cells to facilitate efficient responses to environmental chemical signals.

Finding the Brain in the Nose

Olfaction is fundamentally distinct from other sensory modalities. Natural odor stimuli are complex mixtures of volatile chemicals that interact in the nose with a receptor array that, in rodents, is built from more than 1,000 unique receptors. These interactions dictate a peripheral olfactory code, which in the brain is transformed and reformatted as it is broadcast across a set of highly interconnected olfactory regions. Here we discuss the problems of characterizing peripheral population codes for olfactory stimuli, of inferring the specific functions of different higher olfactory areas given their extensive recurrence, and of ultimately understanding how odor representations are linked to perception and action. We argue that, despite the differences between olfaction and other sensory modalities, addressing these specific questions will reveal general principles underlying brain function.

Differential Sensing of Saccharides Based on an Array of Fluorinated Benzosiloxaborole Receptors

Fluorinated benzosiloxaboroles–silicon congeners of benzoxaboroles, were synthesized and tested as molecular receptors for mono- and disaccharides. The receptors differed in the Lewis acidity of the boron center as well as in the number of potential binding sites. The calculated stability constants indicated different binding affinity of benzosiloxaborole derivatives towards selected saccharides, enabling their classification using a receptor array-based sensing. Unique fluorescence fingerprints were created on the basis of competitive interactions of the studied receptors with both Alizarin Red S (ARS) and tested saccharide molecules. Detailed chemometric analysis of the obtained fluorescence data (based on partial least squares-discriminant analysis and hierarchical clustering analysis) provided the differential sensing of common saccharides, in particular the differentiation between glucose and fructose. In addition, DFT calculations were carried out to shed light on the binding mechanism under different pH conditions.

Syndecan-1 Is Critical in ARF6-Dependent Macropinocytosis Driven By KRAS Mutation in the Pathophysiology of Multiple Myeloma

Syndecan-1 (SDC1), also known as CD138, is a member of integral membrane heparin sulfate proteoglycans constantly expressed in plasma cells (PCs) and a primary diagnostic marker for human multiple myeloma (MM). We here further define new functions of SDC1 in the MM pathobiology. Firstly, flow cytometry and qRT-PCR analysis showed that SDC1 is expressed at relatively higher levels in AMO-1, U266, OPM2, H929, MM1S, and MM1R MM cells when compared with JJN3, RPMI 8226, and ANBL6 MM cells. SDC1 levels are comparable in paired MM cell lines sensitive or resistant to current anti-MM therapies including lenalidomide, pomalidomide, and bortezomib. Significantly increased SDC1 mRNA levels in advanced MM stages (p<0.05) were further correlated with elevated soluble SDC1 protein levels in patient serum by ELISA. As expected, higher soluble SDC1 was also detected in culture media (CM) from MM cell lines with higher mRNA levels. Next, the effects of SDC1 were studied by SDC1 knockout (KO) in OPM2, JJN3 and H929 cells via CRISPR/Ca9 gene modification, followed by RNA-Seq analysis. Neglectable shed SDC1 in CM of all SDC1 KO MM cells confirm null SDC1 expression. Expression of anti-apoptosis gene BCL2L1, cell cycle genes (CCND1, CCND2), and transcription factor RELA gene were decreased in SDC1 KO vs control MM cells. Permanent SDC1 KO cells were eventually derived, indicating additional SDC1 function besides its role in MM cell growth and survival. KEGG pathway analysis associated with genes downregulated following SDC1 KO showed biological processes (BPs) enrichment in ECM-receptor interaction (hsa04512; p< 0.001), cell adhesion molecules (hsa04514; p<0.001), focal adhesion (hsa04510; p<0.001), cytokine-cytokine receptor interaction (hsa04060; p=0.005), chemokine signaling pathway ( hsa04062; p=0.006), gap junction (hsa04540; p=0.002), axon guidance (hsa04360; p=0.016), JAK-STAT signaling pathway (hsa04630; p=0.026), lysosome (hsa04142; p=0.047).Specifically, IL-21R, related to JAK-STAT signaling pathway and cytokine-cytokine receptor interaction, was significantly decreased in SDC1 KO MM cells, as validated by qRT-PCR and human receptor array analysis. IL-21R contains the common cytokine-receptor gamma-chain shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, indicating potential cross-talks between MM cells and surrounding immune cells via SDC1. Since its natural ligand IL-21 is mainly secreted by non-myeloma bone marrow (BM) accessory cells, SDC1 could also modulate interactions between myeloid lineages and MM cells via IL-21/IL-21R circuit in the BM microenvironment. Of note, other key MM antigens, i.e., CD38, BCMA, SLAMF7 were affected at mRNA levels in SDC1 KO vs control MM cells. Moreover, human receptor array data showed decreased expression in Flt-3L, DR6, Endoglin, GITR, HVEM, IL-2RG, IL-17RA, IL-21R, PECAM-1, PDGFRB, RAGE, Trappin-2 and µPAR in SDC1 KO MM cells. BPs through GO analysis in these downregulated receptors were cell activation (GO:0001775), cell surface receptor signaling pathway (GO:0007166), and immune system process (GO:0002370). KEGG analysis showed that those receptors molecular were enriched in cytokine-cytokine receptor interaction pathway (KEGG:04060).Consistent with RNA seq data, µPAR, an important factor of ARF6-dependent trafficking, was also found significantly downregulated in SDC1 KO MM cells. Since ARF6 activation regulates macropinocytosis, an essential metabolic pathway fueling Ras-driven cancer cells, these data suggest that SDC1 may involve in ARF6-dependent macropinocytosis in MM cells. ARF6 is induced by KRAS mutation, we thus checked macropinocytic index in KRAS-mutated MM cell lines. Increased macropinocytosis occur in KRAS-mutated MM cells (KMS28-BM, MM1S, MM1R) compared with KRAS WT OPM2 and KMS12-BM. Importantly, macropinocytosis was inhibited following SDC1 depletion in KRAS-mutated MM cells, indicating that SDC1 critically mediates KRAS-driven macropinocytosis in MM cells. These data highlight the requirements for SDC1 to mediate nutrient-scavenging macropinocytosis in MM cells, most prominently harboring KRAS-mutation. Taken together, our results identify new functions of SDC1 which are crucial to enhance myeloma cell fitness and adaptation to various conditions in the BM milieu, thereby further supporting SDC1 targeted immunotherapy in MM. Disclosures Munshi: Celgene: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Adaptive: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Metal-assisted selective recognition of biothiols by a synthetic receptor array

A synergistic combination of a deep cavitand host, fluorophore guests and transition metal ions can be used to sense small molecule thiols of biological interest with good efficiency and selectivity in complex aqueous media.

Site selective reading of epigenetic markers by a dual-mode synthetic receptor array

Arrayed, self-folding deep cavitands form a fluorescence displacement assay system for the site-selective sensing of post-translationally modified (PTM) histone peptides.

An unexpected use of ferrocene. A scanning electrochemical microscopy study of a toll-like receptor array and its interaction with E. coli

Toll-like receptor microarrays were investigated by scanning electrochemical microscopy with enhanced contrast from using ferrocene derivatives.

Receptor arrays optimized for natural odor statistics

Natural odors typically consist of many molecules at different concentrations. It is unclear how the numerous odorant molecules and their possible mixtures are discriminated by relatively few olfactory receptors. Using an information theoretic model, we show that a receptor array is optimal for this task if it achieves two possibly conflicting goals: (i) Each receptor should respond to half of all odors and (ii) the response of different receptors should be uncorrelated when averaged over odors presented with natural statistics. We use these design principles to predict statistics of the affinities between receptors and odorant molecules for a broad class of odor statistics. We also show that optimal receptor arrays can be tuned to either resolve concentrations well or distinguish mixtures reliably. Finally, we use our results to predict properties of experimentally measured receptor arrays. Our work can thus be used to better understand natural olfaction, and it also suggests ways to improve artificial sensor arrays.