Biology subjects

Coates, C.

Publications and source records attributed to Coates, C..

6 recordsLinked to original sources

bioRxiv2025

Single kinetochores execute an ordered series of molecular events as the Spindle Assembly Checkpoint is silenced

The Spindle Assembly Checkpoint (SAC) delays anaphase onset until all kinetochores are stably attached to microtubules, thus promoting error-free chromosome segregation. Multiple molecular events are implicated in SAC silencing, including removal of phospho-marks, protein (un)binding, and structural reorganisation of the kinetochore - but we currently lack a quantitative map of how these events unfold through time. Here, we use the levels of the checkpoint protein MAD2 to create a pseudo-timeline of SAC silencing at single kinetochores. We demonstrate how silencing proceeds through an ordered series of molecular events where MAD2-Spindly unbinds first and then the KNL1 catalytic platform disassembles, with a pool of active MPS1 retained. Coincidently, the NDC80 ensemble reconfigures in response to high microtubule occupancy. Kinetochores next switch into a mature attachment state that then undergoes gradual further stabilisation through NDC80 tail dephosphorylation. By preventing biorientation, we also define otherwise hidden kinetochore states involved in error correction cycles. This includes a "poised" state which we propose allows for error correction and rapid reactivation of the SAC. These results provide a critical temporal framework for understanding the mechanisms of SAC silencing and error correction at single human kinetochores.

cell biology↗

bioRxiv2025

Squash flowers as microhabitats: the effects of floral temperature and humidity on pollen viability and visitor behavior

Flowers represent intricate microcosms shaped by their chemical and micrometeorological properties. Notable examples include thermogenic flowers that create a warm microclimate for visitors and residents. We document a distinct microclimate within the large flowers of both wild and domesticated squashes (Cucurbita spp.). Unlike thermogenic flowers, squash flower temperatures remain near ambient, but their humidity consistently exceeds ambient levels from bud to senescence, resulting from stomatal and petal transpiration rather than nectar evaporation. Experimentally reducing humidity in greenhouse-grown male squash flowers results in significant pollen tube rupture, directly impacting plant fitness. To explore the role of floral humidity within a broader ecological context, we performed similar humidity manipulations on squash farms to assess impacts on the behavior of their specialist squash bee pollinator (Xenoglossa pruinosa), generalist pollinators (bumblebees, honeybees), and specialist herbivores (cucurbit beetles). Experimentally reduced floral humidity lower visitation frequency by squash bees but have no effect on generalist pollinators. Manipulation of floral humidity did not influence the foraging duration of any pollinators but impacted the residence of male squash bees in wilted flowers compared with unmanipulated flowers. Finally, there was a positive correlation between the dryness of the ambient air and the abundance of squash bees and cucurbit beetles residing in the humid wilted floral chambers. In conclusion, our findings showcase squash flowers as a humid microhabitat that influences reproductive success directly by affecting pollen viability and indirectly by altering interactions with squash bees, their specialist pollinators.

ecology↗

bioRxiv2023

Spatial control of the APC/C ensures the rapid degradation of Cyclin B1

The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is under the control of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The Checkpoint prevents the APC/C from recognizing Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live cell imaging, in vitro reconstitution, biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We further show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an arginine-anchor in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the nucleosome binding motif on Cyclin B1 reduces its interaction with APC/C and delays its degradation, and cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes constitute a platform to promote the efficient interaction between Cyclin B1 and APC/C and ensure the timely degradation of Cyclin B1 and genomic stability.

cell biology↗

bioRxiv2022

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved Fluorescence Correlation Spectroscopy

Measuring the dynamics with which the regulatory complexes of the cell cycle machinery assemble and disassemble is a crucial barrier to our understanding that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of protein-protein interaction kinetics in living cells. Here, we apply Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

cell biology↗

bioRxiv2019

Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding: affinity variants of iGluSnFR termed iGluh, iGlum and iGlul covering a range of Kd-s (5.8 M, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with a Kd of 9.7 M are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluu and iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluh and Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.\n\nSTATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.

biophysics↗

bioRxiv2017

Ultrafast glutamate sensors resolve synaptic short-term plasticity

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to [~]10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 M and 600 M, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.\n\nSignificance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

neuroscience↗