Samuel J. Cooper

Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity

One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. Here, we describe the regulation of cellular shape by signalling systems using a top-down approach. We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. We suggest a simple Boolean model involving the activation of Rac and Rho GTPases in two compartments to explain the basis for all cell shapes in the dataset. Critically, we also generate a probabilistic graphical model to show how cells explore this space in a deterministic, rather than a stochastic, fashion. We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations.

DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.

Apolar and polar transitions drive the conversion between amoeboid and mesenchymal shapes in melanoma cells

Quantitative imaging of single living tumor cells invading three-dimensional collagen matrices is used in tandem with unsupervised computational analysis to characterize melanoma-cell shape space. Melanoma cells can switch between amoeboid and mesenchymal forms via two different routes in shape space—an apolar and a polar route.

Degradation of (La0.6Sr0.4)0.95(Co0.2Fe0.8)O3−δ Solid Oxide Fuel Cell Cathodes at the Nanometer Scale and below

The degradation of intermediate temperature solid oxide fuel cell (ITSOFC) cathodes has been identified as a major issue limiting the development of ITSOFCs as high efficiency energy conversion devices. In this work, the effect of Cr poisoning on (La0.6Sr0.4)0.95(Co0.2Fe0.8)O3-δ (LSCF6428), a particularly promising ITSOFC cathode material, was investigated on symmetrical cells using electrochemical impedance spectroscopy and multiscale structural/chemical analysis by advanced electron and ion microscopy. The systematic combination of bulk and high-resolution analysis on the same cells allows, for the first time, direct correlation of Cr induced performance degradation with subtle and localized structural/chemical changes of the cathode down to the atomic scale. Up to 2 orders of magnitude reduction in conductivity, oxygen surface exchange rate, and diffusivity were observed in Cr poisoned LSCF6428 samples. These effects are associated with the formation of nanometer size SrCrO4; grain boundary segregation of Cr; enhanced B-site element exsolution (both Fe and Co); and reduction in the Fe valence, the latter two being related to Cr substitution in LSCF. The finding that significant degradation of the cathode happens before obvious microscale change points to new critical SOFC degradation mechanisms effective at the nanometer scale and below.

Regioselective deprotection of orthobenzoates for the synthesis of inositol phosphates

Synthetic myo-inositol 1,4,5-triphosphate, Ins(1,4,5)P(3), and myo-inositol 1,3,4,5-tetraphosphate, Ins(1,3,4,5)P(4), continue to be valuable in biological studies. Inositol orthoesters have proved an important class of intermediate to access these compounds. We investigated the ability of steric bulk from a 4-O protecting group to direct DIBAL-H reduction of inositol orthobenzoates to generate the natural Ins(1,4,5)P(3) precursor 2,3,6-O-tribenzyl myo-inositol. Introduction of an equatorial 4-C-methyl group imparts totally selective reduction and we report the synthesis of novel 4-C-methyl-Ins(1,4,5)P(3) and 4-C-methyl-Ins(1,3,4,5)P(4).

RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer

In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population.

A comprehensive model for the proliferation-quiescence decision in response to endogenous DNA damage in human cells

Significance Controlled transitions of human cells between proliferating and nonproliferating states are essential for normal development and tissue homeostasis. To understand how the decision to proliferate is made in response to positive input from growth factors and negative input from the DNA damage response, we have built a mathematical model of the underlying molecular network, based on data from live cell-imaging experiments. Our model suggests that two major cell-cycle transitions are crucial for decision making: the restriction point, which integrates pro- and antiproliferative signals, and the G1/S transition, which temporarily insulates cells from some aspects of the DNA damage response. Together, our model gives mechanistic insight into how cells maintain both sensitivity and robustness to external signals. Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation–quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

Visualizing quantitative microscopy data: History and challenges

Abstract Data visualization is a fundamental aspect of science. In the context of microscopy-based studies, visualization typically involves presentation of the images themselves. However, data visualization is challenging when microscopy experiments entail imaging of millions of cells, and complex cellular phenotypes are quantified in a high-content manner. Most well-established visualization tools are inappropriate for displaying high-content data, which has driven the development of new visualization methodology. In this review, we discuss how data has been visualized in both classical and high-content microscopy studies; as well as the advantages, and disadvantages, of different visualization methods.

Accelerating Live Single-Cell Signalling Studies

The dynamics of signalling networks that couple environmental conditions with cellular behaviour can now be characterised in exquisite detail using live single-cell imaging experiments. Recent improvements in our abilities to introduce fluorescent sensors into cells, coupled with advances in pipelines for quantifying and extracting single-cell data, mean that high-throughput systematic analyses of signalling dynamics are becoming possible. In this review, we consider current technologies that are driving progress in the scale and range of such studies. Moreover, we discuss novel approaches that are allowing us to explore how pathways respond to changes in inputs and even predict the fate of a cell based upon its signalling history and state.

Double perovskite cathodes for proton-conducting ceramic fuel cells: are they triple mixed ionic electronic conductors?

Abstract 18O and 2H diffusion has been investigated at a temperature of 300 °C in the double perovskite material PrBaCo2O5+δ (PBCO) in flowing air containing 200 mbar of 2H216O. Secondary ion mass spectrometry (SIMS) depth profiling of exchanged ceramics has shown PBCO still retains significant oxygen diffusivity (~1.3 × 10−11 cm2s−1) at this temperature and that the presence of water (2H216O), gives rise to an enhancement of the surface exchange rate over that in pure oxygen by a factor of ~3. The 2H distribution, as inferred from the 2H216O− SIMS signal, shows an apparent depth profile which could be interpreted as 2H diffusion. However, examination of the 3-D distribution of the signal shows it to be nonhomogeneous and probably related to the presence of hydrated layers in the interior walls of pores and is not due to proton diffusion. This suggests that PBCO acts mainly as an oxygen ion mixed conductor when used in PCFC devices, although the presence of a small amount of protonic conductivity cannot be discounted in these materials.