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Macromolecular crowding affects the physicochemistry of the cytoplasm and, in turn, microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range (a process that we refer to as 'homeocrowding') and discuss possible mechanisms for achieving this. Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules.

A genetically encodable, FRET-based sensor enables quantitative measurements of macromolecular crowding in living bacterial and mammalian cells. Macromolecular crowding in cells influences processes such as folding, association and diffusion of proteins and polynucleic acids. Direct spatiotemporal readout of crowding would be a powerful approach for unraveling the structure of the cytoplasm and determining the impact of excluded volume on protein function in living cells. Here, we introduce a genetically encodable fluorescence resonance energy transfer (FRET) sensor for quantifying macromolecular crowding and discuss our application of the sensor in bacterial and mammalian cells.

DNA is more than a carrier of genetic information: It is a highly versatile structural motif for the assembly of nanostructures, giving rise to a wide range of functionalities. In this regard, the structure programmability is the main advantage of DNA over peptides, proteins, and small molecules. DNA amphiphiles, in which DNA is covalently bound to synthetic hydrophobic moieties, allow interactions of DNA nanostructures with artificial lipid bilayers and cell membranes. These structures have seen rapid growth with great potential for medical applications. In this Review, the current state of the art of the synthesis of DNA amphiphiles and their assembly into nanostructures are first summarized. Next, an overview on the interaction of these DNA amphiphiles with membranes is provided, detailing on the driving forces and the stability of the interaction. Moreover, the interaction with cell surfaces in respect to therapeutics, biological sensing, and cell membrane engineering is highlighted. Finally, the challenges and an outlook on this promising class of DNA hybrid materials are discussed. DNA nanotechnology is rapidly developing and the derived structures are combined with living systems. Thereby, DNA amphiphiles play an important role. In this Review, the interactions of this class of nucleic acids with membranes and the resulting applications are highlighted. Finally, future research directions of these emerging DNA‐based materials are discussed.

The enantioselective addition of water to olefins in an aqueous environment is a common transformation in biological systems, but was beyond the ability of synthetic chemists. Here, we present the first examples of a non-enzymatic catalytic enantioselective hydration of enones, for which we used a catalyst that comprises a copper complex, based on an achiral ligand, non-covalently bound to (deoxy)ribonucleic acid, which is the only source of chirality present under the reaction conditions. The chiral β-hydroxy ketone product was obtained in up to 82% enantiomeric excess. Deuterium-labelling studies demonstrated that the reaction is diastereospecific, with only the syn hydration product formed. So far, this diastereospecific and enantioselective reaction had no equivalent in conventional homogeneous catalysis. Nature frequently shows exquisite control over reactions both of water and in water. Here, the enantioselective conjugate addition of water to an enone — a reaction that has no equivalent in conventional homogeneous catalysis — is catalysed by a copper complex of an achiral ligand that is non-covalently bound to DNA.

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here, we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells, we observe drastic changes in organellar volume, leading to crowding on the micrometer scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.

Hydrophobic hydrocarbons are absorbed by cell membranes. The effects of hydrocarbons on biological membranes have been studied extensively, but less is known how these compounds affect lipid phase separation. Here, we show that pyrene and pyrene-like hydrocarbons can dissipate lipid domains in phase separating giant unilamellar vesicles at room temperature. In contrast, related aromatic compounds left the phase separation intact, even at high concentration. We hypothesize that this behavior is because pyrene and related compounds lack preference for either the liquid-ordered (Lo) or liquid-disordered (Ld) phase, while larger molecules prefer Lo, and smaller, less hydrophobic molecules prefer Ld. In addition, our data suggest that localization in the bilayer (depth) and the shape of the molecules might contribute to the effects of the aromatic compounds. Localization and shape of pyrene and related compounds are similar to cholesterol and therefore these molecules could behave as such.

The intracellular environment is crowded with macromolecules that influence biochemical equilibria and biomacromolecule diffusion. The incorporation of such crowding in synthetic cells would be needed to mimic the biochemistry of living cells. However, only a few methods provide crowded artificial cells, moreover providing cells with either heterogeneous size and composition or containing a significant oil fraction. Therefore, a method that generates monodisperse liposomes with minimal oil content and tunable macromolecular crowding using polydimethylsiloxane (PDMS)‐based microfluidics is presented. Lipid stabilized water‐in‐oil‐in‐water emulsions that are stable for at least several months and with a high macromolecular crowder concentration that can be controlled with the external osmolality are formed. A crucial feature is that the oil phase can be removed using high flow conditions at any point after production, providing the highly crowded liposomes. Genetically encoded macromolecular crowding sensors show that the high level of macromolecular crowding in the emulsions is fully retained throughout the generation of minimal‐oil lipid bilayers. This modular and robust platform will serve the study of biochemistry under physiologically relevant crowding conditions. A method to achieve monodisperse liposomes with high internal macromolecular crowding is presented. Double emulsions obtained from microfluidics are highly robust and reproducible and can be shrunk to desired crowding through osmosis. Crucially, the oil phase can be removed at any point using a centrifugation step to yield liposomes, while maintaining crowding as verified with a crowding sensor.

Supramolecular assembly has been used to design and create new proteins capable of performing biomimetic functions in complex environments such as membranes and inside living cells.

Abstract Proteins assemble into a tremendous variety of dynamic and functional structures. Sensitive measurements directly in cells with a high spatiotemporal resolution are needed to distinguish these different assemblies. Here, we demonstrate precise and continuous monitoring of cytoplasmic protein self-assemblies and their structural transitions. Intermolecular FRET with both the donor and acceptor protein at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We measure different assembly structures, transient intermediate states’ kinetics, and assembly formation resolved in space and time. Specifically, the method recapitulates that i) the mutant Huntingtin exon1 (mHttex1) protein first forms low-FRET and presumably less ordered assemblies in yeast and human cells, which develop into high-FRET aggregates, ii) the chaperone DNAJB6b prevents low-FRET mHttex1 assemblies, yet coassembles with mHttex1 aggregates, and iii) FUS’ condensates have mutation-dependent nanoscopic structures. FACS measurements allow assembly measurement in a high-throughput manner crucial for screening efforts, while fluorescence microscopy provides spatiotemporally-resolved measurements on the single-condensate level during a cell’s lifetime to assess the biological consequences. Implementation in other native or non-native proteins could provide insight into many studies involving protein condensation or aggregation. Competing Interest Statement The authors have declared no competing interest.