Biology subjects

Bieling, P.

Publications and source records attributed to Bieling, P..

11 recordsLinked to original sources

bioRxiv2026

A self-organized signaling hierarchy patterns the cortex during cell repair

Cells generate dynamic patterns of Rho GTPase activation to direct the subsequent patterning of Rho GTPase effectors needed to remodel the cell cortex during processes ranging from cell division to cell repair. To understand how such patterns arise, we used live cell imaging, time-resolved Rho GTPase manipulations, and a novel computational tool to study the spatiotemporal dynamics of Rho, Cdc42 and several downstream Cdc42 targets in wounded Xenopus laevis oocytes. We find that the characteristic wound-induced segregation of Rho and Cdc42 activity into concentric zones is followed by polarization of the Cdc42 zone such that Toca-1 progressively concentrates at the back of the Cdc42 zone while Arp2/3, cofilin, cortactin, and the Rho GAP p190RhoGAP progressively concentrate at the front of the Cdc42 zone, where it overlaps the Rho zone. Remarkably, the juxtaposition of Rho activity to Cdc42 is required for the polarization of p190RhoGAP, while p190RhoGAP is responsible for establishing the boundary between the Cdc42 and Rho zones. The results indicate that the characteristic segregation of the Rho and Cdc42 zones, as well as the polarization of the Cdc42 zone, arise from cortical self-organization. Further, these findings reveal a simple mechanism for hierarchical establishment of cortical patterns: recruitment of new proteins to regions of signaling compartment overlap.

cell biology↗

bioRxiv2026

Molecular orchestration of global actin remodelling by INF2

The actin cytoskeleton rapidly reorganizes in response to intracellular calcium signals, driving cellular morphogenesis and wound healing. Among actin regulators, the formin INF2 uniquely mediates the "Calcium-mediated Actin Reset" (CaAR) reaction, orchestrating transient and global actin remodeling upon calcium influx. Excessive INF2 activity is linked to kidney and neuronal diseases, underscoring the need for its tight control. Combining live cell imaging with single molecule tracking, biochemistry and structural analysis we discover that INF2 activity is tightly controlled by two interlinked mechanisms: canonical intramolecular autoinhibition and binding of the INF2 N-terminus to the side of actin filaments. Side-binding limits actin elongation and supports re-establishment of autoinhibition. Disruption of this negative feedback prolongs INF2 activity, affecting plasma membrane organization and repair as well as transcriptional control. Our findings uncover a novel product-inhibition mechanism that limits INF2 function and offer important insight into disease mechanisms linked to actin dysregulation.

cell biology↗

bioRxiv2025

Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of CP and VASP

Cell migration on rigid surfaces employs flat protrusions termed lamellipodia, which constitute the prime model system for branched actin filament networks that generate pushing forces for membrane movement. Branched actin filaments are nucleated by the Arp2/3 complex and play vital roles in various cell biological processes, such as organelle trafficking, phagocytosis and autophagy. Here we utilize genome editing to explore the functional connections between the actin monomer-binding protein profilin (Pfn), the filament nucleating Arp2/3 complex, its co-factor heterodimeric capping protein (CP) and the Ena/VASP family of actin filament polymerases in lamellipodial actin assembly. Individual and combinations of knockouts show that Pfn counters Ena/VASP but promotes Arp2/3 complex activity, while Ena/VASP and CP mutually antagonize each other. Notably, while Pfn is important for Arp2/3 complex activity irrespective of Ena/VASP, sensitivity of CP to Pfn removal is lost in the absence of Ena/VASP. Our findings establish Pfn as master regulator of Arp2/3 complex-dependent actin network formation, which differentially regulates VASP and its antagonizer CP. Finally, mathematical modeling of our data suggest that Ena/VASP and CP compete for binding at the lamellipodial edge, likely contributing to their functional antagonism at this subcellular site. This work provides critical insights into the molecular logic of branched actin network assembly in membrane protrusion and cellular force generation.

cell biology↗

bioRxiv2024

Differential interference with actin-binding protein function by acute Cytochalasin B

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding {beta}-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

cell biology↗

bioRxiv2023

Transient structural properties of the Rho GDP-dissociation inhibitor

Rho GTPases are master spatial regulators of the cytoskeleton that control a wide range of cellular processes. Their inactivation by removal from cellular membranes involves the stepwise formation of a stable complex with guanine nucleotide dissociation inhibitors (RhoGDIs), for which process the RhoGDI N-terminus is indispensable. The formation of this interface has been thought to emerge from an intrinsically disordered state of RhoGDI in its free, apo form. Here we use tailored solution NMR analyses, molecular dynamics simulations, and biochemical essays to pinpoint the site-specific structural features of full-length RhoGDI1 before and after binding its GTPase client Cdc42. In contrast to the current mechanistic understanding, a diverse set of NMR data unequivocally shows that the structural properties of the GDI N-terminus seen in crystal structures of the complex with GTPases already exist as largely preformed features in free, apo GDI. Even more interestingly, the required structural properties are imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. Lastly, upon Cdc42 binding, the flexibility of the N-terminus and its secondary-structural propensities are not largely abrogated. These observations change the textbook picture of the mechanism of membrane extraction of the GTPase. Rather than a disorder-to-order transition upon binding, an active role of the N-terminus with differentially preformed structural properties, suitably modulated by the specific surrounding along the multi-step process, seems required to leverage the intricate and highly selective extraction process.

biophysics↗

bioRxiv2023

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments

The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pi release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a molecular backdoor. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why Pi escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated Pi release after polymerization and filaments with drastically shortened ADP-Pi caps. This demonstrates that the Pi release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Pi and Mg2+ at the nucleotide-binding site. Our results provide the molecular basis for Pi release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.

biochemistry↗

bioRxiv2022

Mechanism of threonine ADP-ribosylation of F-actin by a Tc toxin

Tc toxins deliver toxic enzymes into host cells by a unique injection mechanism. One of these enzymes is TccC3, an ADP-ribosyltransferase from Photorhabdus luminescens. Once TccC3 is translocated into the target cell, the enzyme ADP-ribosylates actin, resulting in clustering of the actin cytoskeleton and ultimately cell death. Here, we combine biochemistry, solution and solid-state NMR spectroscopy and cryo-EM to show in atomic detail how TccC3 modifies actin. We find that the ADP-ribosyltransferase does not bind to G-actin but interacts with two consecutive actin subunits of F-actin. The binding of TccC3 to F-actin occurs via an induced-fit mechanism that facilitates access of NAD+ to the nucleotide binding pocket. The following nucleophilic substitution reaction results in the transfer of ADP-ribose to threonine-148 of F-actin. We demonstrate that this site-specific modification of F-actin prevents its interaction with depolymerization factors, such as cofilin, which impairs actin network turnover and leads to steady actin polymerization. Our findings reveal in atomic detail a new mechanism of action of a bacterial toxin through specific targeting and modification of F-actin.

biochemistry↗

bioRxiv2021

The molecular mechanism of load adaptation by branched actin networks

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Unexpectedly, force slows the rate of filament nucleation by promoting the interaction of nucleation promoting factors with actin filament ends, limiting branch formation. This inhibition is countered by an even larger force-induced drop in the rate of filament capping, resulting in a shift in the balance between nucleation and capping that increases network density. Remarkably, the force dependence of capping is identical to that of filament elongation because they require the same size gap to appear between the filament and load for insertion. These results provide direct evidence that Brownian Ratchets generate force and govern the load adaptation of branched actin networks.

biophysics↗

bioRxiv2021

A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal "tentacle" extensions. Deletion of this {beta} tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.

biochemistry↗

bioRxiv2019

Profilin and formin constitute a pacemaker system for robust actin filament growth

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

biochemistry↗

bioRxiv2019

Extraction of active RhoGTPases by RhoGDI regulates spatiotemporal patterning of RhoGTPases

The RhoGTPases are characterized as membrane-associated molecular switches cycling between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these tools, we identified pools of active and inactive RhoGTPase associated with the membrane, showed that RhoGDI can actively extract both inactive and active RhoGTPases, and that the extraction of active RhoGTPase contributes to their spatial regulation around wounds. In contrast to the textbook model of the RhoGTPase cycle, these results indicate that RhoGDI actively contributes to spatiotemporal patterning by removing active RhoGTPases from the plasma membrane.

cell biology↗