Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

International audienceAsgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures

Actin network architecture can ensure robust centering or sensitive decentering of the centrosome

International audienceThe orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture

Recycling of the actin monomer pool limits the lifetime of network turnover

International audienceIntracellular organization is largely mediated by actin turnover. Cellular actin networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called "dynamic steady state," allows cells to sense and adapt to their environment. However, how structural stability can be maintained during the constant turnover of a limited actin monomer pool is poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled by an actin comet in a microwell containing a limited amount of components. We used the speed and the size of the actin comet tails to evaluate the system's monomer consumption and its lifetime. We established the relative contribution of actin assembly, disassembly, and recycling for a bead movement over tens of hours. Recycling mediated by cyclase-associated protein (CAP) is the key step in allowing the reuse of monomers for multiple assembly cycles. ATP supply and protein aging are also factors that limit the lifetime of actin turnover. This work reveals the balancing mechanism for long-term network assembly with a limited amount of building blocks

Microtubules under mechanical pressure can breach dense actin networks

International audienceThe crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment

Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis

International audienceAbstract Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota

New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation

International audienceCo-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial’s structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner’s dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice

Friction patterns guide actin network contraction

International audienceThe shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile actomyosin networks in vitro to study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we used glass or lipids surface micropatterning to control the initial shape of the network. We adapted the concentration of Nucleating Promoting Factor on each surface to induce the assembly of actin networks of similar densities and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction, both the active and resistive forces are essential to direct the actin network deformation

data_sheet_1_New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation.docx

<p>Co-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial’s structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner’s dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice.</p