Biology subjects

Kerssemakers, J.

Publications and source records attributed to Kerssemakers, J..

10 recordsLinked to original sources

bioRxiv2026

Division of Synthetic Cells Using a Genomically Encoded One-Protein Divisome

Synthetic biology is emerging as a powerful tool to understand life by building minimal synthetic cells whose functions are encoded within their genome. An essential function of cells is abscission, the final step of cell division that breaks the last connection between two emerging daughter cells. Abscission is challenging to reconstitute as it is energetically unfavorable to rearrange the cell membrane. Here, we demonstrate a minimal one-component abscission machinery for synthetic cells using the bacterial protein dynamin A (DynA), which is uniquely capable of driving scission inside membrane necks. Cell-free expression of this synthetic cell module is complicated by the large size of DynA (137 kDa) and the presence of a hydrophobic lipid-binding loop within its structure. We overcome these challenges by expressing DynA inside lipid vesicles with a high fraction of negatively charged lipids. We find that DynA enriches at the bridges of dumbbell-shaped vesicles, where it drives membrane scission and full division. The ability to monitor DynA dynamics through genomic encoding further provides hints about the mechanism of abscission including cooperative enrichment at the necks and constriction of the necks. Finally, we demonstrate that the smaller (71 kDa) dynamin D1 domain alone exerts the same function, which is beneficial for future integration with other synthetic cell modules since its much smaller gene size reduces the burden on the synthetic genome. This autonomously operating abscission machinery presents the first example of a self-dividing synthetic cell, marking an important step towards constructing a synthetic cell that is able to sustainably replicate.

synthetic biology↗

bioRxiv2026

Charge and steric hindrance in the glycocalyx govern membrane interactions

The outer leaflet of the plasma membrane - the cells interface with exogenous biomolecules and particles - is largely electrostatically neutral and highly viscous, conditions that are generally unfavorable for fusion of external membrane-bound objects such as liposomes. Yet living cells efficiently interact and fuse with charged liposomes, revealing a discrepancy between synthetic and biological membranes. Here, we identify the glycocalyx, the carbohydrate-rich layer that coats cells, as the component that explains this mismatch. Using synthetic membranes reconstituted with an artificial glycocalyx, we resolve how electrostatics and steric hindrance regulate the interactions of cationic liposomes with synthetic membranes. Charged glycolipids are found to be sufficient to convert non-fusogenic neutral membranes into fusion-competent membranes. In contrast, large polymers in the glycocalyx suppress fusion. Steric hindrance acts primarily at the docking stage, while fusion proceeds through a long-lived hemifusion intermediate. Strikingly, these large differences occur without measurable changes in membrane viscosity or phase state, demonstrating that glycocalyx-mediated control is independent of the membrane mechanics. Selected glycocalyx components can also promote endocytic budding and cargo internalization. We thus show that the glycocalyx is a tunable physicochemical regulator of liposome docking, fusion, and internalization. The findings provide a framework for understanding how biological agents and therapeutics engage cell membranes and they offer key design principles for synthetic cells.

biophysics↗

bioRxiv2026

Lipid-conjugated DNA enables on-demand delivery of lipids and proteins to synthetic cells

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

synthetic biology↗

bioRxiv2024

A microfluidic platform for extraction and analysis of bacterial genomic DNA

Bacterial cells organize their genomes into a compact hierarchical structure called the nucleoid. Studying the nucleoid in cells faces challenges because of the cellular complexity while in vitro assays have difficulty in handling the fragile megabase-scale DNA biopolymers that make up bacterial genomes. Here, we introduce a method that overcomes these limitations as we develop and use a microfluidic device for the sequential extraction, purification, and analysis of bacterial nucleoids in individual microchambers. Our approach avoids any transfer or pipetting of the fragile megabase-size genomes and thereby prevents their fragmentation. We show how the microfluidic system can be used to extract and analyze single chromosomes from B. subtilis cells. Upon on-chip lysis, the bacterial genome expands in size and DNA-binding proteins are flushed away. Subsequently, exogeneous proteins can be added to the trapped DNA via diffusion. We envision that integrated microfluidic platforms will become an essential tool for the bottom-up assembly of complex biomolecular systems such as artificial chromosomes.

biophysics↗

bioRxiv2023

Direct observation of a crescent-shape chromosome in Bacillus subtilis

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genomic information. Direct visualization of the 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 intrinsic shape of the bacterial chromosome in real-time in live Bacillus subtilis cells. We show that the chromosome exhibits a crescent shape 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.

microbiology↗

bioRxiv2022

CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CTCF generates topologically associating domains (TADs)2-4 that play key roles in gene regulation and recombination during development and disease1,5-8. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unknown9. To address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, as well as to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted, but unexpectedly is dependent on DNA tension. Moreover, CTCF regulates cohesins loop extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, where the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

biophysics↗

bioRxiv2022

Quantitative analysis of surface wave patterns of Min proteins

The Min protein system is arguably the best-studied model systems for biological pattern formation. It exhibits pole-to-pole oscillations in E. coli bacteria as well as a variety of surface wave patterns in in vitro reconstitutions. Such Min surface wave patterns pose particular challenges to quantification as they are typically only semi-periodic and non-stationary. Here, we present a methodology for quantitatively analyzing such Min patterns, aiming for reproducibility, user-independence, and easy usage. After introducing pattern-feature definitions and image-processing concepts, we present an analysis pipeline where we use autocorrelation analysis to extract global parameters such as the average spatial wavelength and oscillation period. Subsequently, we describe a method that uses flow-field analysis to extract local properties such as the wave propagation velocity. We provide descriptions on how to practically implement these quantification tools and provide Python code that can directly be used to perform analysis of Min patterns.

biophysics↗

bioRxiv2021

Directing Min protein patterns with advective bulk flow

The Min proteins constitute the best-studied model system for pattern formation in cell biology. We theoretically predict and experimentally show that the propagation direction of in vitro Min protein patterns can be controlled by a hydrodynamic flow of the bulk solution. We find downstream propagation of Min wave patterns for low MinE:MinD concentration ratios, upstream propagation for large ratios, but multistability of both propagation directions in between. Whereas downstream propagation can be described by a minimal model that disregards MinE conformational switching, upstream propagation can be reproduced by a reduced switch model, where increased MinD bulk concentrations on the upstream side promote protein attachment. Our study demonstrates that a differential flow, where bulk flow advects protein concentrations in the bulk, but not on the surface, can control surface-pattern propagation. This suggests that flow can be used to probe these features and to constrain mathematical models for pattern-forming systems.

biophysics↗

bioRxiv2019

mPies: a novel metaproteomics tool for the creation of relevant protein databases and automatized protein annotation

Metaproteomics allows to decipher the structure and functionality of microbial communities. Despite its rapid development, crucial steps such as the creation of standardized protein search databases and reliable protein annotation remain challenging. To overcome those critical steps, we developed a new program named mPies (metaProteomics in environmental sciences). mPies allows the creation of protein databases derived from assembled or unassembled metagenomes, and/or public repositories based on taxon IDs, gene or protein names. For the first time, mPies facilitates the automatization of reliable taxonomic and functional consensus annotations at the protein group level, minimizing the well-known protein inference issue which is commonly encountered in metaproteomics. mPies workflow is highly customizable with regards to input data, workflow steps, and parameter adjustment. mPies is implemented in Python 3/Snakemake and freely available on GitHub: https://github.com/johanneswerner/mPies/.

bioinformatics↗

bioRxiv2017

Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

Condensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

biophysics↗