Biology subjects

Dekker, C.

Publications and source records attributed to Dekker, C..

At least 55 records · Page 3Linked to original sources

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

Transport receptor occupancy in Nuclear Pore Complex mimics

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess Kap-centric NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

biophysics↗

bioRxiv2021

A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

synthetic biology↗

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↗

bioRxiv2021

ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by Molecular Dynamics simulations which also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

biophysics↗

bioRxiv2021

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC

The Cdv proteins constitute the cell-division system of the Crenarchaea, in a protein machinery that is closely related to the ESCRT system of eukaryotes. The CdvB paralog CdvB1 is believed to play a major role in the constricting ring that is the central actor in cell division in the crenarchaea. Here, we present an in vitro study of purified CdvB1 from the crenarchaeon M. sedula with a combination of TEM imaging and biochemical assays. We show that CdvB1 self-assembles into filamentous polymers that are depolymerized by the action of the Vps4-homolog ATPase CdvC. Using liposome flotation assays, we show that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Interestingly, we find that the polymerization and the membrane binding are mutually exclusive properties of the protein. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

biochemistry↗

bioRxiv2021

Probing nanomotion of single bacteria with graphene drums

Motion is a key characteristic of every form of life1. Even at the microscale, it has been reported that colonies of bacteria can generate nanomotion on mechanical cantilevers2, but the origin of these nanoscale vibrations has remained unresolved3,4. Here, we present a novel technique using drums made of ultrathin bilayer graphene, where the nanomotion of single bacteria can be measured in its aqueous growth environment. A single E. coli cell is found to generate random oscillations with amplitudes of up to 60 nm, exerting forces of up to 6 nN to its environment. Using mutant strains, we are able to pinpoint the bacterial flagella as the main source of nanomotion. By real-time tracing of changes in nanomotion upon administering antibiotics, we demonstrate that graphene drums can perform antibiotic susceptibility testing with single-cell sensitivity. These findings deepen our understanding of processes underlying cellular dynamics, and pave the way towards high throughput and parallelized rapid screening of the effectiveness of antibiotics in bacterial infections with graphene devices.

biophysics↗

bioRxiv2021

SMC complexes can traverse physical roadblocks bigger than their ring size

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, how ever, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion. One-sentence summaryHuge DNA-bound roadblocks can be incorporated into SMC-extruded DNA loops, pointing to a nontopological mechanism for loop extrusion.

biophysics↗

bioRxiv2021

Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to rewind peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer. One-sentence summaryThis paper presents proof-of-concept experiments and simulations of a nanopore-based approach to sequencing individual proteins.

biophysics↗

bioRxiv2021

Condensin-driven loop extrusion on supercoiled DNA

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, e.g., the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping is stimulated by positive supercoiled DNA where it preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects all nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

biophysics↗

bioRxiv2021

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations

Many strategies have been pursued to trap and monitor single proteins over time in order to detect the molecular mechanisms of these essential nanomachines. Single protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection based on ion currents. Here we present the Nanopore Electro-Osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with sub-millisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtraps ability to sensitively distinguish proteins based on size and shape, and discriminate nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

biophysics↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

synthetic biology↗

bioRxiv2021

Reconstitution of ultrawide DNA origami pores in liposomes for transmembrane transport of macromolecules

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed new methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective as dextran molecules with a diameter up to 22 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modelling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, synthetic biology, to drug delivery.

biophysics↗

bioRxiv2020

Resolving the step size in condensin-driven DNA loop extrusion identifies ATP binding as the step-generating process

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ~200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

biophysics↗

bioRxiv2020

FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in bacterial cell division

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments1,2. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear2-5. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a novel combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

microbiology↗

bioRxiv2020

DNA sequence-directed cooperation between nucleoid-associated proteins

Nucleoid associated proteins (NAPs) are a class of highly abundant DNA binding proteins in bacteria and archaea. While the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding to the bacterial chromosome and stabilising higher-order nucleoprotein complexes. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding-site arrangements. We find that spatial organisation of the protein binding sites can govern the higher-order architecture of the nucleoprotein complexes. Based on sequence arrangement the complexes differed in their global shape and compaction, as well as the extent of FIS and H-NS binding. Our observations highlight the important role the DNA sequence plays in driving structural differentiations within the bacterial chromosome.

biophysics↗

bioRxiv2020

Phase separation induced by cohesin SMC protein complexes

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

biophysics↗

bioRxiv2020

Bulk-surface coupling reconciles Min-protein pattern formation in vitro and in vivo

Self-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we show that the Min protein dynamics on the membrane crucially depend on bulk gradients normal to the membrane. A theoretical analysis shows that in vitro patterns at low bulk height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger bulk height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work qualitatively resolves the Min systems in vivo/in vitro conundrum and provides important insights on the mechanisms underlying protein patterns in bulk-surface coupled systems.

biophysics↗