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Biology subjects

Colin, A.

Publications and source records attributed to Colin, A..

8 recordsLinked to original sources

Actin pool controls the scaling of dynamic actin networks

Scaling is a fundamental property of living systems, allowing cells to match the size of their internal components to their volume. The size and shape of cells depend largely on the actin cytoskeleton, whose dynamic self-assembling structures are central to this adaptation. Yet how they self-assemble in a volume-dependent manner remains unresolved. To address this question, we used a biomimetic system consisting of beads propelled by actin polymerization in microwells of different volumes, which allowed us to work with a limited and defined number of components. This approach enabled the first reconstitution, from purified proteins, of dynamic cytoskeletal structures that adapt to the volume of their environment. The scalability of these structures depends on how strongly they deplete monomers from a shared pool, and tuning the disassembly rate is sufficient to shift their scaling regime. Scaling principles also hold for multiple competing structures, with greater competition imposing stronger constraints on the monomer pool. Together, these results establish a mechanistic framework in which a structure's capacity to adapt is set by how much it draws down shared resources as it grows, a principle that may extend to other intracellular structures that scale with the cells that build them.

biochemistry↗

Fabrication of microcompartments with controlled size and shape for encapsulating active matter

In all living systems, the cytoplasm is separated from the external environment by membranes. This confinement imposes spatial constraints on the self-organization of internal components, filaments and organelles. While reconstituted systems are instrumental for understanding fundamental biological principles, traditional experiments often utilize volumes vastly larger than actual cells. In recent studies, water-in-oil droplets or giant unilamellar vesicles have been widely used to impose confinement. However, these compartments present imaging challenges and make precise protein content control difficult. To address these limitations, we have developed versatile microwells that are straightforward to implement, compatible with different types of imaging, suitable for long-term experiments, and capable of generating large amounts of data. These microwells are compatible with several surface treatments and a wide range of experimental techniques (such as micropatterning) making them a powerful tool for answering key questions in cell biology. We present here a detailed protocol of the fabrication of the microwells as well as characterization of the method to ensure quality throughout the manufacturing process. These microwells support various cytoskeleton-based processes including actin polymerization, dynamic steady-state actin networks, and composite actin-microtubule networks. More broadly, they can be used to encapsulate and study over time any kind of active matter.

biochemistry↗

Filament transport supports contractile steady states of actin networks.

In all eukaryotic cells, the actin cytoskeleton is maintained in a dynamic steady-state. Actin filaments are continuously displaced from cell periphery, where they assemble, towards the cells center, where they disassemble. Despite this constant flow and turnover, cellular networks maintain their overall architecture constant. How such a flow of material can support dynamic yet steady cellular architectures remains an open question. To investigate the role of myosin-based forces in contractile steady-states of actin networks, we used a reconstituted in vitro system based on a minimal set of purified proteins, namely actin, myosin and actin regulators. We found that, contrary to previous bulk experiments, when confined in microwells, the actin network could self-organize into ordered arrangements of contractile bundles, flowing continuously without collapsing. This was supported by three-dimensional fluxes of actin filaments, spatially separated yet balancing each other. Unexpectedly, maintaining these fluxes did not depend on filament nucleation or elongation, but solely on filament transport. Ablation of the contractile bundles abolished the flux balance and led to network collapse. These findings demonstrate that the dynamic steady state of actin networks can be sustained by filament displacement and recirculation, independently of filament assembly and disassembly. Significance StatementCellular structures continuously self-renew, with new material constantly being added and removed while maintaining overall structural stability. This is particularly true for the actin cytoskeleton, whose components are continuously assembled, displaced, and reassembled. Understanding this process is fundamental to uncovering how cells regulate their architecture and adapt to stimuli. Here, we reconstitute an in vitro actomyosin network capable of contracting steadily over time without collapsing, relying solely on myosin-based transport. These findings demonstrate that a minimal system consisting of actin and molecular motors can effectively recapitulate the ability of actin networks to self-organize into stable yet dynamic architectures.

biophysics↗

Balancing limited resources in actin networks competition

In cells, multiple actin networks coexist in a dynamic manner. These networks compete for a common pool of actin monomers and actin-binding proteins. Interestingly, this competition does not result in the mere survival of the more consuming networks. Moreover, the co-existence of networks with various strengths is key to cell adaption to external changes. However, a comprehensive view of how these networks coexist in this competitive environment, where resources are limited, is still lacking. To address this question, we used a reconstituted system, in closed microwells, consisting of beads propelled by actin polymerization or micropatterns functionalized with lipids capable of initiating polymerization close to a membrane. This system enabled us to build dynamic actin architectures, competing for a limited pool of proteins, over a period of hours. We demonstrated the importance of protein turnover for the coexistence of actin networks, showing it ensures resource distribution between weak and strong networks. However, when competition becomes too intense, turnover alone is insufficient, leading to a selection process that favors the strongest networks. Consequently, we emphasize the importance of competition strength, which is defined by the turnover rate, the amount of available protein, and the number of competing structures. More generally, this work illustrates how turnover allows biological populations with various competition strengths to coexist despite resource constraints. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/612098v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@370343org.highwire.dtl.DTLVardef@1b75a2forg.highwire.dtl.DTLVardef@81e55dorg.highwire.dtl.DTLVardef@1f97452_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cell biology↗

Friction patterns guide actin network contraction

The 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 acto-myosin 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 micropatterned actin nucleation promoting factors on glass or on a lipid bilayer. We adapted their concentrations 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. Significance statementCell shape changes are controlled by complex sets of mechanical forces of various origins. Numerous studies have been dedicated to the role of active forces, originating from molecular motors and filament polymerization, but much less is known about the guiding role of resistive forces. Here we show that a non-uniform distribution of friction forces between a contracting acto-myosin network and its underlying substrate can direct its deformation as it contracts. Our results suggest that the contribution of resistive forces, such as anchoring forces but also less specific viscous forces along cell surface, can be as significant as those of active forces in driving network deformation and should be considered in mechanical models describing the regulation of cell shape and movements.

biophysics↗

Recycling limits the lifetime of actin turnover

Intracellular organization is largely mediated by the actin turnover. Cellular actin networks consume matter and energy to sustain their dynamics, while maintaining their appearance. This behavior, called dynamic steady state, enables 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 using actin bead motility in a compartment with a limited amount of monomer. We used the speed and the size of the actin comet tails to evaluate the systems 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 proteins is the key step in allowing the reuse of monomers for multiple assembly cycles. Energy 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.

cell biology↗

Two different cell-cycle processes determine the timing of cell division in Escherichia coli

Cells must control the cell cycle to ensure that key processes are brought to completion. In Escherichia coli, it is controversial whether cell division is tied to chromosome replication or to a replication-independent inter-division process. A recent model suggests instead that both processes may limit cell division with comparable odds in single cells. Here, we tested this possibility experimentally by monitoring single-cell division and replication over multiple generations at slow growth. We then perturbed cell width, causing an increase of the time between replication termination and division. As a consequence, replication became decreasingly limiting for cell division, while correlations between birth and division and between subsequent replication-initiation events were maintained. Our experiments support the hypothesis that both chromosome replication and a replication-independent inter-division process can limit cell division: the two processes have balanced contributions in non-perturbed cells, while our width perturbations increase the odds of the replication-independent process being limiting.

systems biology↗

Centering based on active diffusion in mouse oocytes is non-specific

The mechanism for nucleus centering in mouse oocytes results from a gradient of actin-positive vesicles. By microinjecting oil droplets and fluorescent beads, we analyze the consequences of the gradient of activity on transport of exogenous tracer particles of different sizes. We also use optical tweezers to probe rheological properties of the cytoplasm. We find that the gradient activity induces a general centering force, akin to an effective pressure gradient, leading to centering of oil droplets with velocities comparable to nuclear ones. High temporal resolution measurements reveal that passive particles, larger than 1{micro}m, experience the activity gradient by a biased diffusion towards the cell center. Unexpectedly, this general and size dependent mechanism is maintained in Meiosis I but contrasted by a further process that specifically off-centers the spindle. These antagonizing processes depend on myosin activity, thus we reconcile how the same molecular actors can have two opposite functions (centering versus off-centering).

cell biology↗