Biology subjects

Davis, S. J.

Publications and source records attributed to Davis, S. J..

6 recordsLinked to original sources

bioRxiv2018

Temperature but not the circadian clock determines nocturnal carbohydrate availability for growth in cereals

The circadian clock is considered a key target for crop improvement because it controls metabolism and growth in Arabidopsis. Here, we show that the clock gene EARLY FLOWERING 3 (ELF3) controls vegetative growth in Arabidopsis but not in the cereal crop barley. Growth in Arabidopsis is determined by the degradation of leaf starch reserves at night, which is controlled by ELF3. The vegetative growth of barley, however, is determined by the depletion of leaf sucrose stores through an exponential kinetics, presumably catalyzed by the vacuolar sucrose exporter SUCROSE TRANSPORTER 2 (SUT2). This process depends on the sucrose content and the nighttime temperature but not on ELF3. The regulation of starch degradation and sucrose depletion in barley ensures efficient growth at favorable temperature as stores become exhausted at dawn. On cool nights, however, only the starch degradation rate is compensated against low nighttime temperatures, whereas the sucrose depletion rate is reduced. This coincides with reduced biomass in barley but not in Arabidopsis after growth in consecutive cool nights. The sucrose depletion metabolism determines growth in the cereal crops barley, wheat, and rice but is not generally conserved in monocot species and is not a domestication-related trait. Therefore, the control of growth by endogenous (clock) versus external factors (temperature) is species-specific and depends on the predominant carbohydrate store. Our results give new insights into the physiology of growth in cereals and provide a basis for studying the genetics and evolution of different carbohydrate stores and their contribution to plant productivity and adaptation.\n\nSignificance StatementThe circadian clock controls growth in the model plant Arabidopsis thaliana by regulating the starch degradation rate so that reserves last until dawn. This prevents nocturnal starvation until photosynthesis resumes. The cereal crops barley, wheat and rice, however, predominantly consume sucrose instead of starch as carbohydrate source. We find that carbohydrate supply from sucrose at night is regulated by enzyme kinetics and night-time temperature, but not the circadian clock. We postulate that the regulation of growth depends on the predominant carbohydrate store, where starch degradation is controlled by endogenous cues (clock) and sucrose depletion by external factors (temperature). These differences in the regulation of carbohydrate availability at night may have important implications for adapting crops yields to climate change.

plant biology↗

bioRxiv2018

Hydrodynamic trapping measures the interaction between membrane-associated molecules

How membrane proteins distribute and behave on the surface of cells is determined by the molecules interaction potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. We here present how a method we call hydrodynamic trapping can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding from a lipid membrane. The interaction potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this to study four representative proteins, with different height-to-width ratios and protein properties; from the globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The obtained data illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins as well as underline the general applicability of the method.

biophysics↗

bioRxiv2018

Photoperiod sensing of the circadian clock is controlled by ELF3 and GI

ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well-studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock-controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light-dark cycles even under diurnal conditions. Consequently, clock-mediated photoperiod-responsive growth and development is completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod-insensitive crops by adjusting the allelic combination of these two key genes.\n\nOne sentence summaryELF3 and GI are essential for circadian clock mediated photoperiod sensing.

plant biology↗

bioRxiv2018

Reconstitution of immune cell interactions in free-standing membranes

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. It is important, therefore, to be able to elucidate molecular processes occurring at these interfaces. However, the detailed investigation of each components contribution to the formation and regulation of the contact is hampered by the complexity of cellular composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially for using advanced imaging technology. One approach to circumventing these problems is to establish in vitro systems that faithfully mimic immune cell interactions, incorporating complexity that can be dialled-in as needed. Here, we present an in vitro system making use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we begin to investigate the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and the intracellular rearrangements that accompany the initiation of signalling in T cells. The model system presented here is expected to be widely applicable.\n\nSummary StatementImmune cell-cell interactions are reconstituted in free-standing vesicles wherein spatiotemporal aspects of immune synapse formation can be investigated.

immunology↗

bioRxiv2017

The stoichiometric signature of Rhodopsin-family G protein coupled receptors

Whether Rhodopsin-family G protein-coupled receptors (GPCRs) form dimers is highly controversial, with much data both for and against emerging from studies of mostly individual receptors. The types of large-scale comparative studies from which a consensus could eventually emerge have not previously been attempted. Here, we sought to determine the stoichiometric \"signatures\" of 60 GPCRs expressed by a single human cell-line using orthogonal bioluminescence resonance energy transfer-based and single-molecule microscopy assays. We observed that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appeared to be monomeric. Mapped onto the entire family the analysis predicted that fewer than 20% of the ~700 Rhodopsin- family receptors form dimers. The clustered distribution of Rhodopsin-family dimers, and a striking correlation between receptor stoichiometry and GPCR family-size that we also identified, suggested that evolution has tended to favor the lineage expansion of monomers rather than dimers.\n\nOne Sentence SummaryAnalysis of 71 GPCRs from a single cell reveals the strong tendency of Rhodopsin-family receptors to exist as monomers rather than form dimers.

biochemistry↗

bioRxiv2017

Constraining CD45 exclusion at close-contacts provides a mechanism for discriminatory T-cell receptor signalling

The T-cell receptor (TCR) triggers the elimination of pathogens and tumors by T lymphocytes. In order for this to avoid damage to the host, the receptor has to discriminate between thousands of peptide ligands presented by each host cell. Exactly how the TCR does this is unknown. In resting T-cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over kinases expressed at the cell surface. When agonist peptides are presented to the TCR by major histocompatibility complex (MHC) proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering occurs, leading to TCR phosphorylation. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we develop and test a quantitative treatment of receptor triggering reliant only upon TCR dwell-time in phosphatase-depleted cell-cell contacts constrained in area by cell topography. Using the model and experimentally-derived parameters, we find that ligand discrimination is possible but that it depends crucially on individual contacts being 400 nm in diameter or smaller, i.e. the size generated by microvilli. The model not only correctly predicts the relative signaling potencies of known agonists and non-agonists, but achieves this in the absence of conventional, multi-step kinetic proof-reading. Our work provides a simple, quantitative and predictive molecular framework for understanding why TCR triggering is so selective and fast, and reveals that for some receptors, cell topography crucially influences signaling outcomes.\n\nSignificance statementOne approach to testing biological theories is to determine if they are predictive. A simple, theoretical treatment of TCR triggering suggests that ligand discrimination by the receptor relies on just two physical principles: (1) the time TCRs spend in cell-cell contacts depleted of large tyrosine phosphatases; and (2) constraints on contact size imposed by T cells using finger-like protrusions to interrogate their targets. The theory not only allows agonistic and non-agonistic TCR ligands to be distinguished but predicts the relative signalling potencies of agonists with remarkable accuracy. This suggests that the theory captures the essential features of receptor triggering.

immunology↗