Biology subjects

Litschel, T.

Publications and source records attributed to Litschel, T..

7 recordsLinked to original sources

bioRxiv2025

3D printing cytoskeletal networks: ROS-induced filament severing leads to surge in actin polymerization

The cytoskeletal protein actin forms a spatially organized biopolymer network that plays a central role in many cellular processes. Actin filaments continuously assemble and disassemble, enabling cells to rapidly reorganize their cytoskeleton. Filament severing accelerates actin turnover, as both polymerization and depolymerization rates depend on the number of free filament ends. Here, we use light to control actin severing in vitro by locally generating reactive oxygen species (ROS) with photosensitive molecules such as fluorophores. We see that ROS sever actin filaments, which increases actin polymerization in our experiments. However, beyond a certain threshold, excessive severing leads to the disassembly of actin networks, allowing us to selectively remove actin structures. Our experimental data is supported by simulations using a kinetic model of actin polymerization, which helps us understand the underlying dynamics. In cells, ROS are known to regulate the actin cytoskeleton, but the molecular mechanisms are poorly understood. Here we show that, in vitro, ROS directly affect actin reorganization.

biophysics↗

bioRxiv2024

Mechanistic studies of autophagic cargo recruitment and membrane expansion through in vitro reconstitution

Autophagy is a highly conserved catabolic pathway to remove deleterious cytosolic material to maintain cellular homeostasis and cell survival. Upon autophagy induction, a unique double-membraned structure, called a phagophore, forms and expands into a cup shape to engulf these cytosolic substrates. ATG8 proteins are covalently conjugated to autophagic membranes by lipidation of phosphatidylethanolamine (PE) and are thought to localise on both sides of the phagophore membrane. ATG8 conjugated on the inner membrane of the phagophore recruits autophagy cargo receptors, such as p62. To recapitulate events on the inner membrane, we used giant unilamellar vesicles (GUVs) as a model membrane and encapsulated proteins of interest inside GUVs, thus generating a membrane platform to which ATG8 proteins could be localised on the inner leaflet of the vesicles. We reconstituted WIPI2b-directed and cargo-directed ATG8 lipidation inside the GUVs and revealed distinct roles of WIPI2b and p62 in initiating the ATG conjugation cascade. Furthermore, we showed that p62 or p62 droplets were recruited to the inner membrane of the GUVs though interaction with membrane-bound ATG8s. Using a bead-based membrane expansion assay, we demonstrated a redistribution and local enrichment of membrane-bound ATG8s across the membrane upon interaction with p62 and p62 droplets. Our study provides novel model systems to investigate the interactions on the inner membrane of the phagophore and reveals fundamental molecular insights into phagophore membrane bending. This process is directed by ATG8-cargo interaction, during which cargo receptors concentrate ATG8 proteins on the inner surface of the phagophore membrane.

biochemistry↗

bioRxiv2023

Membrane-induced 2D phase separation of focal adhesion proteins

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol.

cell biology↗

bioRxiv2023

Salactin, a dynamically unstable actin homolog in Haloarchaea

Across the domains of life, actin homologs are integral components of many essential processes such as DNA segregation, cell division, and cell shape determination. Archaea genomes, like those of bacteria and eukaryotes, also encode actin homologs, but much less is known about these proteins in vivo dynamics and cellular functions. We identified and characterized the function and dynamics of Salactin, an actin homolog in the hypersaline archaeon Halobacterium salinarum. Despite Salactins homology to bacterial MreB proteins, we find it does not function as a MreB ortholog in H. salinarum. Rather, live-cell imaging revealed that Salactin forms dynamically unstable filaments that grow and shrink out of the cell poles. Like other dynamically unstable polymers, Salactin monomers add at the growing filament end and its ATP-bound critical concentration is substantially lower than the ADP-bound form. When H. salinarums chromosomal copy number becomes limiting under low phosphate growth conditions, cells lacking Salactin show perturbed DNA distributions. Taken together, we propose that Salactin is part of a previously unknown chromosomal segregation apparatus required during low-ploidy conditions.

microbiology↗

bioRxiv2020

Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture

How complex actin network architectures arise and coexist in membrane-enclosed cell environment remains unknown. By encapsulating actin and crosslinking proteins -actinin and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement and its size can lead to formation of complex actin structures, including rings and asters at GUV peripheries and centers. Strikingly, we find that the materials properties of the aster structures depend on the ratio of the relative concentrations of -actinin and fascin, and we demonstrate that this results from -actinin and fascin sorting into separate domains in the structures. We complement our experiments with molecular simulations that capture the spontaneous formation of competing network architectures; these provide a microscopic view of the dynamics and delineate the molecular features that promote sorting. We propose that the observed boundary-imposed effect on protein sorting is a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.

biophysics↗

bioRxiv2020

Reconstitution of contractile actomyosin rings in vesicles

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

synthetic biology↗

bioRxiv2019

Microfluidic trapping of vesicles reveals membrane-tension dependent FtsZ cytoskeletal re-organisation

The geometry of reaction compartments can affect the outcome of chemical reactions. Synthetic biology commonly uses giant unilamellar vesicles (GUVs) to generate cell-sized, membrane-bound reaction compartments. However, these liposomes are always spherical due to surface area minimization. Here, we have developed a microfluidic chip to trap and reversibly deform GUVs into rod- or cigar-like shapes, including a constriction site in the trap mimicking the membrane furrow in cell division. When we introduce into these GUVs the bacterial tubulin homologue FtsZ, the primary protein of the bacterial Z ring, we find that FtsZ organization changes from dynamic rings to elongated filaments upon GUV deformation, and that these FtsZ filaments align preferentially with the short GUV axis, in particular at the membrane neck. In contrast, pulsing Min oscillations in GUVs remained largely unaffected. We conclude that microfluidic traps are a useful tool for deforming GUVs into non-spherical membrane shapes, akin to those seen in cell division, and for investigating the effect of confinement geometry on biochemical reactions, such as protein filament self-organization.

synthetic biology↗