DNA is the carrier of all cellular genetic information and increasingly used in nanotechnology. Quantitative understanding and optimization of its functions requires precise experimental characterization and accurate modeling of DNA properties. A defining feature of DNA is its helicity. DNA unwinds with increasing temperature, even for temperatures well below the melting temperature. However, accurate quantitation of DNA unwinding under external forces and a microscopic understanding of the corresponding structural changes are currently lacking. Here we combine single-molecule magnetic tweezers measurements with atomistic molecular dynamics and coarse-grained simulations to obtain a comprehensive view of the temperature dependence of DNA twist. Experimentally, we find that DNA twist changes by ΔTw(T) = (−11.0 ± 1.2)°/(°C·kbp), independent of applied force, in the range of forces where torque-induced melting is negligible. Our atomistic simulations predict ΔTw(T) = (−11.1 ± 0.3)°/(°C·kbp), in quantitative agreement with experiments, and suggest that the untwisting of DNA with temperature is predominantly due to changes in DNA structure for defined backbone substates, while the effects of changes in substate populations are minor. Coarse-grained simulations using the oxDNA framework yield a value of ΔTw(T) = (−6.4 ± 0.2)°/(°C·kbp) in semi-quantitative agreement with experiments.

Cell polaritythe morphological and functional differentiation of cellular compartments in a directional manneris required for processes such as orientation of cell division, directed cellular growth and motility. How the interplay of components within the complexity of a cell leads to cell polarity is still heavily debated. In this Review, we focus on one specific aspect of cell polarity: the non-uniform accumulation of proteins on the cell membrane. In cells, this is achieved through reaction-diffusion and/or cytoskeleton-based mechanisms. In reaction-diffusion systems, components are transformed into each other by chemical reactions and are moving through space by diffusion. In cytoskeleton-based processes, cellular components (i.e. proteins) are actively transported by microtubules (MTs) and actin filaments to specific locations in the cell. We examine how minimal systemsin vitro reconstitutions of a particular cellular function with a minimal number of componentsare designed, how they contribute to our understanding of cell polarity (i.e. protein accumulation), and how they complement in vivo investigations. We start by discussing the Min protein system from Escherichia coli, which represents a reaction-diffusion system with a well-established minimal system. This is followed by a discussion of MT-based directed transport for cell polarity markers as an example of a cytoskeleton-based mechanism. To conclude, we discuss, as an example, the interplay of reaction-diffusion and cytoskeleton-based mechanisms during polarity establishment in budding yeast.

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-mNeonGreen and four Cdc42-sfGFP 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.

Establishing cell polarity is vital for cells, as it is required for cell division, directed growth and secretion, and motility. A well-studied model organism for polarity establishment is Saccharomyces cerevisiae: here the small Rho-type GTPase Cdc42 exits the cytoplasm and accumulates in one spot on the cell membrane, marking the site of bud emergence. Due to redundancy and interconnection within the regulatory network surrounding Cdc42, the molecular mechanisms driving Cdc42 accumulation continue to be a subject of intense debate. In this study, we utilize a bulk in vitro GTPase assay to examine the GTPase cycle of Cdc42 in combination with two of its effectors - the GDP/GTP exchange factor (GEF) Cdc24 and GTPase activating protein (GAP) Rga2. We find that Cdc24's GEF activity scales non-linearly with its concentration, which might be linked to Cdc24 di- or oligomerisation alleviating its autoinhibition. In contrast to Cdc24, Rga2 has an order of magnitude weaker GTPase cycle boosting effect which saturates at μM concentrations. Notably, Cdc24 combined with Rga2 leads to a large synergy in boosting Cdc42's GTPase activity, which we hypothesise to be caused by the elevation of the Rga2 activity through Cdc24. Our data exemplifies a novel synergy within the regulatory network of Cdc42. This synergy contributes to efficient regulation of Cdc42's GTPase cycle over a wide range of cycling rates, enabling cells to resourcefully establish polarity. As Cdc42 is highly conserved among eukaryotes, we suspect the GEF-GAP synergy to be a general regulatory property in other eukaryotes.