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Roos, W. H.

Publications and source records attributed to Roos, W. H..

14 recordsLinked to original sources

MEMBRANE PORES ACT AS SELF-RESEALING LIPID SCRAMBLASES

Biological membranes continuously experience leaflet lipid imbalances during growth, lipid synthesis, and vesicle fusion. To alleviate such imbalances and prevent these asymmetries from compromising membrane integrity, cells rely on lipid scramblases - fast and non-specific lipid channel proteins. Here we show that lipid number asymmetry alone is sufficient to drive spontaneous formation of transient hydrophilic pores that function as self-resealing lipid scramblases. Using giant unilamellar vesicles, living cells, and coarse-grained molecular dynamics simulations, we demonstrate that fusion-induced excess lipids in one leaflet lowers membrane edge tension, generating size-selective pores whose size and lifetime scale with the magnitude of asymmetry. Below a critical threshold, these pores reseal spontaneously; above it, membranes collapse. Strikingly, pore opening enables rapid, non-selective lipid translocation between leaflets, dissipating the asymmetry that nucleates the pore and thereby promoting their own closure. Cholesterol buffers moderate imbalances through spontaneous flip flop before pore formation, whereas pore-mediated lipid scrambling relieves the remaining asymmetry and restores cholesterols initial distribution. Our findings identify transient lipid pores as an intrinsic, protein-independent mechanism that couples membrane destabilization to self-repair, providing a universal physical principle for membrane homeostasis during growth, remodelling, and early cellular evolution.

biophysics↗

Dimeric Alix nucleates ESCRT-III CHMP4 polymerization

Alix is a key adaptor protein of the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery. Although Alix-mediated recruitment of the ESCRT-III subunit CHMP4 is well established, the molecular mechanisms underlying Alix activation and ESCRT-III polymerization remain poorly understood. Here, we present the crystal structure of the dimeric Alix V-domain (Alix-V) in complex with a llama nanobody. Dimerization is mediated by domain swapping, generating an X-shaped flexible conformation. We demonstrate that Alix forms dimers in vivo and provide evidence for the recruitment of dimeric Alix to plasma membrane repair sites and the cytokinetic midbody. Notably, a mutation disrupting Alix dimerization impairs plasma membrane repair. Furthermore, high-speed AFM experiments reveal that dimeric Alix, but not the monomeric form, nucleates CHMP4B filament polymerization. Our data establish that dimeric Alix is the active form responsible for nucleating two ESCRT-III CHMP4 filaments, whose geometry creates a platform for the recruitment of downstream ESCRT-III components required to assemble the active membrane remodeling and fission machinery.

biochemistry↗

A nested shell structure coordinates enzyme communication in pyruvate oxidation

The pyruvate dehydrogenase complex (PDHc)1 links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2, a process essential for life2,3. PDHc is formed by structural proteins (E3-binding protein, E3BP)4-7, enzymatic subunits (E1, E2, E3)4,6, and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites6,8. Although numerous details regarding pyruvate oxidation steps have been elucidated9, the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico, in vitro, and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a [~]40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies10. Our findings provide structural context for the textbook "link reaction"11, building on decades of biochemical knowledge, are transferable to functional aspects of other -ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/724543v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1615612org.highwire.dtl.DTLVardef@159bc8dorg.highwire.dtl.DTLVardef@69f8c1org.highwire.dtl.DTLVardef@14a83a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Impact of modification of envelope proteins on the mechanical properties of HIV virus-like particles

The mechanical interactions between virus-like particles and host cells may offer targets for new viral treatments and vaccines with modes of action that are independent of the immune system. The physical properties of structures involved govern the particle-cell interactions. While the mechanical properties of virions and mammalian cells have been widely studied, data on virus-like particles are limited. This study aimed to determine the mechanical and morphological properties of HIV-1 virus-like particles with different envelopes. Three HIV-like particles, i.e. GagM + gp150, GagM + gp140HA2tr, and GagM + gp120HA2, were produced by combining the same Gag protein shell with different trimeric glycoprotein envelopes. The particles spring constant, breaking force, and dimensions were determined using atomic force microscopy, and the elastic modulus was quantified using finite element analysis. Spring constant, elastic modulus, and breaking force were higher for GagM + gp140HA2tr and GagM + gp120HA2 than for GagM + gp150. The particle height was smaller for GagM + gp120HA2 than for GagM + gp150 and GagM + gp140HA2tr. Possible mechanisms underlying the increase of the particles stiffness and mechanical strength are the inclusion of the influenza virus HA transmembrane domain in the HIV Env protein, and the lower expression and packing density of Env in GagM + gp140HA2tr and GagM + gp120HA2 compared to GagM + gp150 found previously. Upon confirmation, the proposed mechanisms offer potential to tailor the mechanics of HIV virus-like particles and guide mechanical interactions between VLPs and host cells towards improving vaccines.

biophysics↗

Influence of Substrate on Supported Lipid Bilayers: Membrane Pinning, Stretching, Pores, and Remodelling

Model membranes offer a simplified representation of biomembranes, allowing for controlled structural and functional studies without the inherent complexity of biological systems. Due to the confined space and strong interactions with the support, supported lipid bilayers (SLBs) exhibit markedly distinct properties compared to those of their free-standing counterparts. However, the nano and microscale origins of these differences, structural effects and the related biophysical consequences remain incompletely understood. Using a combination of spectroscopy and micromanipulation techniques, we reveal that SLBs are pinned to the support, creating barriers for lipid diffusion. Furthermore, they are intrinsically stretched and under tension, causing membrane rupture and the formation of stable [~]2 nm aqueous pores that allow the permeation of small hydrophilic molecules. Despite strong adhesion, SLBs can undergo localized shape changes associated with membrane fusion, and membrane nanotubes can be extruded under optical forces. The findings underscore the very distinct properties of model membranes, and we anticipate that they will have important consequences in the field of drug-delivery systems and membrane biophysics.

biophysics↗

Glycan-induced structural activation softens the human papillomavirus capsid for entry through reduction of intercapsomere flexibility

High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigated the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data will help further devising prophylactic strategies against HPV infections.

microbiology↗

Cofilin-Driven Nuclear Deformation Drives Dendritic Cell Migration through the Extracellular Matrix

To mount an adaptive immune response, dendritic cells must process antigens, migrate to lymph nodes and form synapses with T cells. Critical to 3D migration and mechano-sensing is the nucleus, which is the size-limiting barrier for navigation through gaps in the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed, adopting a raison-like shape and enables dendritic cells to overcome the typical 2 - 3-micron pore limit for 3D-migration. We show that the nuclear shape-change is partially attained through reduced cell adhesion, whereas improved migration through extracellular matrix is achieved through reprogramming of the actin cytoskeleton. Specifically we show that cofilin-1 is phosphorylated at serine 41 drives the assembly of a Cofilin-ActoMyosin (CAM)-ring proximal to the nucleus and enhancing migration through 3D collagen gels. In summary, these data describe novel signaling events through which dendritic cells simultaneously deform their nucleus and enhance their migratory capacity; molecular events that may be re-capitulated in other contexts such as wound healing and cancer.

cell biology↗

A new antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

microbiology↗

Lateral membrane organization as target of an antimicrobial peptidomimetic compound

Antimicrobial resistance is one of the leading concerns in medical care. Here we resolve the functional mechanism of the antimicrobial action of the cationic tripeptide AMC-109 by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on the negatively charged plasma membrane of Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates with specificity for negatively charged membranes. Second, by incorporation into the S. aureus membrane the lateral membrane organization is affected, dissolving membrane nanodomains. Domain dissolution affects membrane functions such as protein sorting and cell wall synthesis, and is suggested to cause a loss of resistance of methicillin-resistant S. aureus (MRSA) to methicillin. As the AMC-109 mode of action is similar to the activity of the disinfectant benzalkonium chloride (BAK), a broad applicability, but with low cytotoxicity to human cells, is expected.

biophysics↗

Histone chaperones exhibit conserved functionality in nucleosome remodeling

Chromatin homeostasis mediates some of the most fundamental processes in the eukaryotic cell. In this regard, histone chaperones have emerged as major regulatory factors during DNA replication, repair, and transcription. However, the dynamic nature of these processes has severely impeded their characterization at the molecular level. Here we apply single-molecule probing by fluorescence optical tweezers to follow histone chaperone dynamics in real-time. The molecular action of SET/template-activating factor-I{beta} and nucleophosmin 1--representing the two most common histone chaperone folds--were examined using both nucleosomes and isolated core histones. We show that these chaperones present binding specificity for partially dismantled nucleosomes and are able to recognize and disrupt non-native histone-DNA interactions. Furthermore, we reveal that cytochrome c inhibition of histone chaperones is coupled to chaperone accumulation on DNA-bound histones. Our single-molecule approach shows that despite the drastically different structures of these chaperones, they present conserved modes of action mediating nucleosome remodeling.

biophysics↗

Human ESCRT-III Polymers Assemble on Positively Curved Membranes and Induce Helical Membrane Tube Formation

Endosomal sorting complexes required for transport-III (ESCRT-III) are thought to assemble in vivo inside membrane structures with a negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and conversely how ESCRT-III polymers shape membranes is still unclear. Here, we used human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron microscopy, cryo-electron tomography and high-speed AFM. We show that CHMP4B filaments bind preferentially to flat membranes or to membrane tubes with a positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes, the latter winding around the tubes. Although combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes, they also reshape large unilamellar vesicles into helical membrane tubes with a pipe surface shape. Sub-tomogram averaging reveals that the filaments assemble parallel to the tube axis with some local perpendicular connections, highlighting the particular mechanical stresses imposed by ESCRT-III to stabilize the corkscrew-like membrane architecture. Our results thus underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature of ESCRT-III required for all or selected cellular membrane remodeling processes.

cell biology↗

Synaptotagmin-1 and Doc2b exhibit distinct membrane remodeling mechanisms

While the role of Synaptotagmin-1 in living cells has been described in detail, it remains a challenge to dissect the contribution of membrane remodelling by its two cytoplasmic C2 domains (C2AB) to the Ca2+-secretion coupling mechanism. Here, we study membrane remodeling using pairs of optically-trapped beads coated with SNARE-free synthetic membranes. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. A lipid mixing assay with confocal imaging reveals that at low Syt1 concentrations, no hemifusion is observed. Notably, for similar low concentrations of Doc2b hemifusion does occur. Consistently, both C2AB fragments cause a reduction in the membrane bending modulus, as measured by an AFM-based method. This lowering of the energy required for membrane deformation likely contributes to the overall Ca2+-secretion triggering mechanism by calcium sensor proteins. When comparing symmetrical (both sides) and asymmetrical (one side) presence of protein on the membranes, Syt1 favors an asymmetrical but Doc2b a symmetrical configuration, as inferred from higher tether probabilities and break forces. This provides support for the direct bridging hypothesis for Syt-1, while hinting to possible preference for protein-protein (and not protein-membrane) interactions for Doc2b. Overall, our study sheds new light on the mechanism of Ca2+ induced fusion triggering, which is essential for fundamental understanding of secretion of neurotransmitters and endocrine substances.

biophysics↗

Real-time assembly of an artificial virus elucidated at the single-particle level

While the structure of a variety of viruses has been resolved at atomistic detail, their assembly pathways remain largely elusive. Key unresolved issues in assembly are the nature of the critical nucleus starting particle growth, the subsequent self-assembly reaction and the manner in which the viral genome is compacted. These issues are difficult to address in bulk approaches and are effectively only accessible by tracking the dynamics of assembly of individual particles in real time, as we show here. With a combination of single-molecule techniques we study the assembly into rod-shaped virus-like particles (VLPs) of artificial capsid polypeptides, de-novo designed previously. Using fluorescence optical tweezers we establish that oligomers that have pre-assembled in solution bind to our DNA template. If the oligomer is smaller than a pentamer, it performs one-dimensional diffusion along the DNA, but pentamers and larger oligomers are essentially immobile and nucleate VLP growth. Next, using real-time multiplexed acoustic force spectroscopy, we show that DNA is compacted in regular steps during VLP growth. These steps, of [~]30 nm of DNA contour length, fit with a DNA packaging mechanism based on helical wrapping of the DNA around the central protein core of the VLP. By revealing how real-time, single particle tracking of VLP assembly lays bare nucleation and growth principles, our work opens the doors to a new fundamental understanding of the complex assembly pathways of natural virus particles.

biophysics↗

One-dimensional sliding assists σ70-dependent promoter binding by Escherichia coli RNA polymerase

The search for a promoter on DNA by RNA polymerase (RNAP) is an obligatory first step in transcription. The role of facilitated diffusion during promoter search has been controversial. Here, we re-assessed facilitated diffusion in promoter search by imaging motions of single molecules of Escherichia coli RNAP {sigma}70 holoenzyme on single DNA molecules suspended between optical traps in a manner that absolutely avoided interactions with surfaces. The assay enabled us to observe unambiguous one-dimensional sliding of RNAP {sigma}70 holoenzyme for thousands of DNA base pairs during promoter search. Analysis of binding kinetics revealed short binding events on nonspecific DNA (0.4 s), intermediate binding events on A/T-rich DNA (1.6 s), and long binding events at or near promoters (>300 s). We estimate a lower bound for the \"diffusion facilitation threshold\" - the RNAP concentration at which three-dimensional search and one-dimensional sliding contribute equally to promoter binding - of 0.2 M RNAP. The results suggest facilitated diffusion occurs in promoter search by RNAP, even at the relatively high, 0.2-0.6 M, concentrations of RNAP in cells.\n\nSignificance statementThe flow of genetic information from DNA to RNA is of central importance to living systems, and it can only start after an RNA polymerase (RNAP) has found a promoter site. But how does this enzyme find promoter sites on DNA in the first place? In recent years, debate on this topic has favored a promoter search mechanism that is dominated by three-dimensional diffusion of RNAP, rather than by one-dimensional sliding of RNAP on DNA. Here, we designed an improved single-molecule assay that unambiguously revealed extensive one-dimensional sliding of RNAP on DNA. Our results imply that, at the RNAP concentrations in living cells, the promoter-search process is facilitated by one-dimensional sliding on DNA.

biophysics↗