Biology subjects

Baldauf, L.

Publications and source records attributed to Baldauf, L..

6 recordsLinked to original sources

bioRxiv2024

Target cell tension regulates macrophage trogocytosis

Macrophages are known to engulf small membrane fragments, or trogocytose, target cells and pathogens, rather than fully phagocytose them. However, little is known about what causes macrophages to choose trogocytosis versus phagocytosis. Here, we report that cortical tension of target cells is a key regulator of macrophage trogocytosis. At low tension, macrophages will preferentially trogocytose antibody-opsonized cells, while at high tension they tend towards phagocytosis. Using model vesicles, we demonstrate that macrophages will rapidly switch from trogocytosis to phagocytosis when membrane tension is increased. Stiffening the cortex of target cells also biases macrophages to phagocytose them, a trend that can be countered by increasing antibody surface density and is captured in a mechanical model of trogocytosis. This work suggests that a distinct molecular pathway for trogocytosis is not required to explain differences in trogocytosis among target cell types and points to a mechanism for target cells to modulate trogocytosis.

biophysics↗

bioRxiv2023

Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/562183v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9764cforg.highwire.dtl.DTLVardef@15b63c7org.highwire.dtl.DTLVardef@f39e83org.highwire.dtl.DTLVardef@2bb63a_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.

synthetic biology↗

bioRxiv2023

Biomimetic actin cortices shape cell-sized lipid vesicles

Animal cells are shaped by a thin layer of actin filaments underneath the plasma membrane known as the actin cortex. This cortex stiffens the cell surface and thus opposes cellular deformation, yet also actively generates membrane protrusions by exerting polymerization forces. It is unclear how the interplay between these two opposing mechanical functions plays out to shape the cell surface. To answer this question, we reconstitute biomimetic actin cortices nucleated by the Arp2/3 complex inside cell-sized lipid vesicles. We show that thin Arp2/3-nucleated actin cortices strongly deform and rigidify the shapes of giant unilamellar vesicles and impart a shape memory on time scales that exceeds the time of actin turnover. In addition, actin cortices can produce finger-like membrane protrusions, showing that Arp2/3-mediated actin polymerization forces alone are sufficient to initiate protrusions in the absence of actin bundling or membrane curving proteins. Combining mathematical modeling and our experimental results reveals that the concentration of actin nucleating proteins, rather than actin polymerization speed, is crucial for protrusion formation. This is because locally concentrated actin polymerization forces can drive a positive feedback loop between recruitment of actin and its nucleators to drive membrane deformation. Our work paints a picture where the actin cortex can either drive or inhibit deformations depending on the local distribution of nucleators. Significance StatementThe cells in our body must actively change shape in order to migrate, grow and divide, but they also need to maintain their shape to withstand external forces during tissue development. Cellular shape control results from an interplay between the plasma membrane and its underlying cortex, a shell composed of crosslinked actin filaments. Using cell-free reconstitution and mathematical modelling, we show that minimal biomimetic actin cortices can mechanically rigidify lipid vesicles while at the same time driving membrane protrusion formation. Our observations suggest that the spatial distribution of actin nucleation determines whether the actin cortex drives or inhibits membrane deformations.

biophysics↗

bioRxiv2022

Reconstituted branched actin networks sense and generate micron-scale membrane curvature

The actin cortex is a complex cytoskeletal machinery which drives and responds to changes in cell shape. It must generate or adapt to plasma membrane curvature to facilitate diverse functions such as cell division, migration and phagocytosis. Due to the complex molecular makeup of the actin cortex, it remains unclear whether actin networks are inherently able to sense and generate membrane curvature, or whether they rely on their diverse binding partners to accomplish this. Here, we show that curvature sensing and generation is an inherent capability of branched actin networks nucleated by Arp2/3 and VCA. We develop a robust method to encapsulate actin inside giant unilamellar vesicles (GUVs) and assemble an actin cortex at the inner surface of the GUV membrane. We show that actin forms a uniform and thin cortical layer when present at high concentration and distinct patches that generate negative membrane curvature at low concentration. Serendipitously, we find that the GUV production method also produces dumbbell-shaped GUVs, which we explain using mathematical modelling in terms of membrane hemifusion of nested GUVs. We find that dendritic actin networks preferentially assemble at the neck of the dumbbells, which possess a micron-range convex curvature that matches the curvature generated by actin patches in spherical GUVs. Minimal dendritic actin networks can thus both generate and sense membrane curvatures, which may help mammalian cells to robustly recruit actin to curved membranes in order to facilitate diverse cellular functions such as cytokinesis and migration. SIGNIFICANCEAnimal cells move, deform and divide using their actin cortex, a thin layer of filamentous proteins that supports the plasma membrane. For these actions, actin must often assemble at curved sections of the membrane, which is widely believed to require the action of dedicated actin- or membrane-bending proteins. Here, we use a bottom-up reconstitution approach to ask whether actin networks are intrinsically able to generate and sense membrane curvature. We show that membrane-nucleated actin cortices can indeed preferentially self-assemble at concave membranes generated by hemifusion of lipid vesicles. This raises intriguing questions about how such curvature recognition works, and whether cells exploit this intrinsic capability of branched actin networks to concentrate actin in specific cortical regions.

biophysics↗

bioRxiv2020

Weak catch bonds make strong networks

Molecular catch bonds are ubiquitous in biology and well-studied in the context of leukocyte extravasion1, cellular mechanosensing2,3, and urinary tract infection4. Unlike normal (slip) bonds, catch bonds strengthen under tension. The current paradigm is that this remarkable ability enables cells to increase their adhesion in fast fluid flows1,4, and hence provides strength-on-demand. Recently, cytoskeletal crosslinkers have been discovered that also display catch bonding5-8. It has been suggested that they strengthen cells, following the strength-on-demand paradigm9,10. However, catch bonds tend to be weaker compared to regular (slip) bonds because they have cryptic binding sites that are often inactive11-13. Therefore, the role of catch bonding in the cytoskeleton remains unclear. Here we reconstitute cytoskeletal actin networks to show that catch bonds render them both stronger and more deformable than slip bonds, even though the bonds themselves are weaker. We develop a model to show that weak binding allows the catch bonds to mitigate crack initiation by moving from low- to high-tension areas in response to mechanical loading. By contrast, slip bonds remain trapped in stress-free areas. We therefore propose that the mechanism of catch bonding is typified by dissociation-on-demand rather than strength-on-demand. Dissociation-on-demand can explain how both cytolinkers5-8,10,14,15 and adhesins1,2,4,12,16-20 exploit continuous redistribution to combine mechanical strength with the adaptability required for movement and proliferation21. Our findings provide a new perspective on diseases where catch bonding is compromised11,12 such as kidney focal segmental glomerulosclerosis22,23, caused by the -actinin-4 mutant studied here. Moreover, catch bonds provide a route towards creating life-like materials that combine strength with deformability24.

biophysics↗

bioRxiv2016

Evolutionary variations in the biofilm-associated protein BslA from the genus Bacillus

BslA is a protein secreted by Bacillus subtilis which forms a hydrophobic film that coats the biofilm surface and renders it water-repellent. We have characterised three orthologues of BslA from Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus as well as a paralogue from B. subtilis called YweA. We find that the three orthologous proteins can substitute for BslA in B. subtilis and confer a degree of protection, whereas YweA cannot. The degree to which the proteins functionally substitute for native BslA correlates with their in vitro biophysical properties. Our results demonstrate the use of naturally-evolved variants to provide a framework for teasing out the molecular basis of interfacial self-assembly.

microbiology↗