Cells grow their boundaries by incorporating newly synthesized lipids into their membranes as well as through fusion of intracellular vesicles. As these processes yield trans-bilayer imbalances in lipid numbers, cells must redistribute lipids across the bilayer to enable sustained growth. Using giant and large unilamellar vesicles (GUVs and LUVs, respectively), we here recapitulate cellular growth and division under various conditions of transmembrane 'flip flop' of lipids. By dynamically monitoring the changes in reduced volume, spontaneous curvature, and area difference of GUVs that grow by fusion of many small LUVs, the morphology of these growing 'synthetic cells' is quantified. We demonstrate, for membranes containing various flip flop-capable molecules, that curvature stresses are relieved, generating more symmetrically sized buds, without significantly compromising the membrane integrity. Further increasing the neck curvature is shown to lead to bud scission. The mechanisms presented here offer insights into cell growth and division, which are important for understanding early protocells and designing synthetic cells that are able to grow and divide.

The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin–DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.

Synthetic cells (SynCells) are artificial constructs designed to mimic cellular functions, offering insights into fundamental biology, as well as promising impact in the fields of medicine, biotechnology, and bioengineering. Achieving a functional SynCell from the bottom up, i.e. by assembling it from molecular components, requires a global collaboration to overcome the many challenges of engineering and assembling life-like modules while addressing biosafety, equity, and ethical concerns in order to guide responsible innovation. Here, we highlight major scientific hurdles, such as the integration of functional modules by ensuring compatibility across diverse synthetic subsystems, and we propose strategies to advance the field.

Human cohesin extrudes DNA into loops and is positioned along the genome by stalling at the human CCCTC-binding factor (CTCF) upon encountering its N-terminal region (NTR). The mechanism underlying this stalling, however, is unresolved. Using single-molecule assays that monitor DNA loop extrusion (LE) in the presence of NTR fragments, we identify two amino acid motifs, YDF and KTYQR, which hinder LE. KTYQR is found to completely block LE activity, while YDF hinders cohesin from completing LE step cycles and converts cohesin into a unidirectional extruder by strengthening the affinity of STAG1 to DNA. We thus identify two distinct NTR motifs that stall LE via different yet synergistic mechanisms, highlighting the multifaceted ways employed by CTCF to modulate LE to shape and regulate genomes.

In September 2023, the Biology and Physics of Prokaryotic Chromosomes meeting ran at the Lorentz Center in Leiden, The Netherlands. As part of the workshop, those in attendance developed a series of discussion points centered around current challenges for the field, how these might be addressed, and how the field is likely to develop over the next 10 years. The Lorentz Center staff facilitated these discussions via tools aimed at optimizing productive interactions. This Perspective article is a summary of these discussions and reflects the state-of-the-art of the field. It is expected to be of help to colleagues in advancing their own research related to prokaryotic chromosomes and inspiring novel interdisciplinary collaborations. This forward-looking perspective highlights the open questions driving current research and builds on the impressive recent progress in these areas as represented by the accompanying reviews, perspectives, and research articles in this issue. These articles underline the multi-disciplinary nature of the field, the multiple length scales at which chromatin is studied in vitro and in and highlight the differences and similarities of bacterial and archaeal chromatin and chromatin-associated processes.

Due to its pivotal role as a regulator of nucleocytoplasmic transport, the structure and dynamic gating mechanism of the nuclear pore complex (NPC) is a subject of immense interest. Here, we report key recent advancements discussed at the Selective Transport Control in Biological and Biomimetic Nanopores meeting (Monte Verità, Switzerland, 2024) that gathered NPC experts from a range of disciplines. Novel insights were reported from cutting-edge super-resolution techniques that enable the direct interrogation of the NPC’s dynamic central transporter; computational models that unravel the mechanisms of the selective barrier; and synthetic NPC mimics as valuable in vitro models for delineating NPC permeability and transport dynamics. Altogether, three major insights were highlighted: (i) the presence of dynamically organised nuclear transport pathways within the NPC, (ii) the role of nuclear transport receptors that enrich and reinforce the NPC’s selective permeability barrier, and (iii) the ability of DNA origami nanostructures to mimic aspects of the NPC with unprecedented precision. Overall, the advancements marked a convergence in our understanding of NPC function by unraveling its dynamic gating mechanism at the nanoscale.

Structural maintenance of chromosomes (SMC) complexes organize the genome via DNA loop extrusion. Although some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, although the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. Apart from expanding by extrusion, loops frequently diffuse and shrink. The findings reveal that SMCs, surprisingly, can switch directions.

Bacterial cells require DNA segregation machinery to properly distribute a genome to both daughter cells upon division. The most common system involved in chromosome and plasmid segregation in bacteria is the ParABS system. A core protein of this system - partition protein B (ParB) - regulates chromosome organization and chromosome segregation during the bacterial cell cycle. Over the past decades, research has greatly advanced our knowledge of the ParABS system. However, many intricate details of the mechanism of ParB proteins were only recently uncovered using in vitro single-molecule techniques. These approaches allowed the exploration of ParB proteins in precisely controlled environments, free from the complexities of the cellular milieu. This review covers the early developments of this field but emphasizes recent advances in our knowledge of the mechanistic understanding of ParB proteins as revealed by in vitro single-molecule methods. Furthermore, we provide an outlook on future endeavors in investigating ParB, ParB-like proteins, and their interaction partners.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins – the key drivers of chromosomal organization – along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

Correction to: Nature Communicationhttps://doi.org/10.1038/s41467-024-47094-x, published online 28 March 2024 The original version of this article contained an error in the “Acknowledgement “section. The original version read “We also acknowledge funding for the work in S.G. lab by the Swiss National Science Foundation (grant number: 310030L_170242).” This has been amended to “We also acknowledge funding for the work in S.G. lab by the Swiss National Science Foundation (grant number: 310030_197770).” This has now been corrected in both the PDF and HTML versions of the Article.

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change ΔL _k of ~−0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.