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Daalman, W. K.-G.

Publications and source records attributed to Daalman, W. K.-G..

7 recordsLinked to original sources

Quantification of GTPase cycling rates of GTPases and GTPase : effector mixtures using GTPase Glo™ assays

In different cellular activities like signal transduction, cell division, and intracellular transportation, small GTPases take on a vital role. Their functioning involves hydrolysing guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article we explain the application of a commercially accessible GTPase assay, known as the GTPase Glo assay by Promega, for the quantitative investigation of GTPase - effector interactions and the interplay between effectors. Basic ProtocolConducting GTPase assays with GTPase : effector protein mixtures using the GTPase Glo assay (Promega). Supporting Protocol 1Analysing GTPase assays to correlate the assay readout (luminescence) to amount of remaining GTP. Supporting Protocol 2Fitting GTPase assay data to obtain GTPase cycling rates.

biochemistry↗

The GEF Cdc24 and GAP Rga2 synergistically regulate Cdc42 GTPase cycling

Cell polarity is a crucial biological process essential for cell division, directed growth, and motility. In Saccharomyces cerevisiae, polarity establishment centers around the small Rho-type GTPase Cdc42, which cycles between GTP-bound and GDP-bound states, regulated by GEFs like Cdc24 and GAPs such as Rga2. To dissect the dynamic regulation of Cdc42, we employed in vitro GTPase assays, revealing inverse concentration-dependent profiles for Cdc24 and Rga2: with increasing concentration, Cdc24s GEF activity is non-linear and oligomerization-dependent, which is possibly linked to the relief of its self-inhibition. In contrast, Rga2s GAP activity saturates, likely due to self-inhibition upon oligomerization. Together, Cdc24 and Rga2 exhibit a strong synergy driven by weak Cdc24 - Rga2 binding. We propose that the synergy stems from Cdc24 alleviating the self-inhibition of oligomeric Rga2. We believe this synergy contributes to efficient regulation of Cdc42s GTPase cycle over a wide range of cycling rates, enabling cells to resourcefully establish polarity. As Cdc42 is highly conserved among eukaryotes, we propose the GEF-GAP synergy to be a general regulatory property in other eukaryotes.

biochemistry↗

A guide to the in vitro reconstitution of Cdc42 GTPase activity and its regulation

Cdc42 is a small Rho-type GTPase and the main regulator of cell division in eukaryotes. It is surrounded by a large network of regulatory proteins. To understand the processes around cell division, in-depth understanding of Cdc42 and its regulation is required. In vitro reconstitutions are a suitable tool for such detailed mechanistic studies, as they allow a high level of control over the conditions and components used and. For these Cdc42 and its regulators need to be expressed, purified, and tested for their activity. There are many methods described for this, but their details, possible difficulties, and points of failure are rarely discussed. This makes in vitro studies on Cdc42 less accessible to scientists that have a background different from biochemistry. We here present our experience with working with Cdc42 in vitro. We describe the recombinant expression and purification behaviour of 12 Cdc42, six Cdc42-mNeonGreenSW and four Cdc42-sfGFPSW constructs in E. coli. We explore Cdc42 dimerisation in vitro and assess its activity using GTPase Glo assays and Flag-pulldown assays. GTPase Glo assays turn out to be a reliable tool to quantitatively asses GTPase activities, wheareas pulldown experiments are more error prone. We find that most Cdc42 constructs, with the exception of those with an N-terminal Twin-Step-tag, show a similar GTPase activity and interaction with the GDP/GTP exchange factor Cdc24. We close with using enterokinase and TEV protease to generate untagged Cdc42. Enterokinase also cuts Cdc42 in an undesired position. TEV protease leads to the desired product, which retains its GTPase activity but shows a reduced Cdc24 interaction. The work presented here acts as a guide for scientists desiring to work with Cdc42 in vitro through describing Cdc42s properties in detail and examining assays that can be used to study its behaviour or act as activity checks. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/538075v3_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18e6ecborg.highwire.dtl.DTLVardef@c068aorg.highwire.dtl.DTLVardef@1816ac5org.highwire.dtl.DTLVardef@188baa5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Epigenetic feedback on noisy expression boosts evolvability

Adapting organisms often face fitness valleys, i.e. barriers imposed by ubiquitous genetic interactions, while optimizing functions. Elucidating mechanisms that facilitate fitness valley traversals is integral to understanding evolution. Therefore, we investigated how protein expression noise, mechanistically decomposed into instant variation and epigenetic inheritance of optimal protein dosage ( transgenerational feedback), shapes the fitness landscape. For this purpose, we combined a minimal model for expression noise with diverse data of Saccharomyces cerevisiae from literature on e.g. expression and fitness to representatively simulate mutational fitness effects. For our proxy of point mutations, which are very often near-neutral, instant dosage variation by expression noise typically incurs a 8.7% fitness loss (17% in essential genes) for non-neutral point mutations. However, dosage feedback mitigates most of this deleterious effect, and additionally extends the time until extinction when essential gene products are underexpressed. Taken together, we consider dosage feedback as a relevant example of Waddingtons canalization: a mechanism which temporarily drives phenotypes towards the optimum upon a genetic mismatch, thereby promoting fitness valley traversal and evolvability. Author summaryGene products frequently interact to generate unexpected phenotypes. This universal phenomenon is known as epistasis, and complicates step-wise evolution to an optimum. Attempts to understand and/or predict how the optimum is found are further compromised by the countless combinations of mutations that are considered by nature, and necessitate the formulation of general rules on how the obstacles that epistasis presents are bridged. To make such a rule as insightful as possible, we reduced cell division to a generation-based model focusing on one protein at a time for reproductive success. Importantly, protein production between divisions is stochastic and we show how the resulting expression noise affects epistasis. After validating the model on experimental fitness landscapes, we combine high-throughput data of budding yeast from multiple sources to make our model predictions on mutational effects on fitness as representative as possible. We find different effects per mutation type: gene duplications have little effect, as genes in our simulated pool are rarely toxic, loss-of-function mutations decrease mutational gains as adaptation progresses, and point mutations permit expression noise to unlock its roles in adaptation. For non-neutral point mutations, noise imposes a sizeable fitness penalty or even induces extinction, which is alleviated by an epigenetic, transgenerational feedback on protein dosage which is never deleterious. Particularly for essential genes, we predict that this effect reduces the obstacles of epistasis and hence significantly increases evolvability, adding to the general rules of evolution.

evolutionary biology↗

Predicting an epistasis-rich genotype-phenotype map with a coarse-grained bottom-up model of budding yeast polarity

Accurate phenotype prediction based on genotypical information has numerous societal applications, such as design of useful crops of cellular factories. However, the prevalence of epistasis, a phenomenon that prevents many biological systems to perform in accordance with the sum of its parts, necessitates modelling the complex path between genotype and phenotype. Defining intermediate levels in this path reduces the complexity of prediction, and may also elucidate the phenotype coupling to other levels by evolution. Inconveniently, the latter requires definitions that maintain biophysical justification from the bottom-up, which conflicts with tractability. By means of a cell growth model, we exemplify a resolution for this conflict by polarization of Cdc42p in budding yeast, a process requiring clustering of active Cdc42p to one zone on the membrane and known to generate ample epistasis. Concretely, our model parsimoniously encompasses constant membrane area growth, stochastic Cdc42p turnover and a simple, justifiable polarity rule we define as the mesotype. Through intuitively interpretable simulations, we describe previously documented, yet puzzling epistasis inside the polarity module. Moreover, we generate evolutionary relevant predictions e.g., on environmental perturbations, which are general enough to apply to other systems. We quantify how poor growth medium can equalize fitness differentials and enables, otherwise very distinct, evolutionary paths. For example, the fitness of the crippled {Delta}bem1 relative to WT can easily be raised from 0.2 to above 0.95. Finally, we can extend our predictions on epistasis to other modules. We determine that modelled epistasis predictions only add predictive value when functional information of the involved modules is included. This inspires a road-map towards modelling the bidirectional genotype-phenotype map for other model systems with abundant interactions, where the intermediate levels reveal targets that evolution can optimize and facilitate a biophysical justifiable incorporation of epistasis. Author summaryEfforts to understand how traits follow from genes facilitate a broad range of applications. For example, crops can be engineered faster to better resist drought, salt and heat stress, and medicines can be better tailored to individuals. Unfortunately, the path from genes to traits can generally involve a complex interplay of hundreds of genes and gene products whose individual contributions can be heavily context-dependent. In this work, we provide the proof-of-concept in a relatively simple system for a road-map towards elucidating this path. We have constructed a cell growth model for budding yeast, only involving simple rules on membrane growth, protein production and centrally, polarity, the process where yeast chooses the future division site. Despite the simplicity, the polarity rule is fully justifiable from underlying biophysics. Model simulations show good accordance with formerly puzzling traits, and also predict the ease with which the environment can change evolutionary paths. While lab conditions may prohibit the emergence of certain polarity mutations, this becomes much more feasible in the wild. The tractable model nature allows us to extrapolate the context dependence of mutational effects beyond polarity, showing that this method for understanding trait generation also helps to elucidate protein evolution.

evolutionary biology↗

Predicting an epistasis-rich genotype-phenotype map with a coarse-grained bottom-up model of budding yeast polarity

Accurate phenotype prediction based on genetic information has numerous societal applications, such as crop design or cellular factories. Epistasis, when biological components interact, complicates modelling phenotypes from genotypes. Here we show an approach to mitigate this complication for polarity establishment in budding yeast, where mechanistic information is abundant. We coarse-grain molecular interactions into a so-called mesotype, which we combine with gene expression noise into a physical cell cycle model. Firstly, we show with computer simulations that the mesotype allows validation of the most current biochemical polarity models by quantitatively matching doubling times. Secondly, the mesotype elucidates epistasis emergence as exemplified by evaluating the predicted mutational effect of key polarity protein Bem1p when combined with known interactors or under different growth conditions. This example also illustrates how unlikely evolutionary trajectories can become more accessible. The tractability of our biophysically justifiable approach inspires a road-map towards bottom-up phenotype modelling beyond statistical inferences.

systems biology↗

Adaptability and evolution of the cell polarization machinery in budding yeast

How can a self-organized cellular function evolve, adapt to perturbations, and acquire new sub-functions? To make progress in answering these basic questions of evolutionary cell biology, we analyze, as a concrete example, the cell polarity machinery of Saccharomyces cerevisiae. This cellular module exhibits an intriguing resilience: it remains operational under genetic perturbations and recovers quickly and reproducibly from the deletion of one of its key components. Using a combination of modeling, conceptual theory, and experiments, we show that multiple, redundant self-organization mechanisms coexist within the protein network underlying cell polarization and are responsible for the modules resilience and adaptability. Based on our mechanistic understanding of polarity establishment, we hypothesize how scaffold proteins, by introducing new connections in the existing network, can increase the redundancy of mechanisms and thus increase the evolvability of other network components. Moreover, our work suggests how a complex, redundant cellular module could have evolved from a more rudimental ancestral form.

evolutionary biology↗