Wenzheng Shi

Dramatic movements characterize rapid assembly of mitotic spindle in prometaphase: chromosomes congress to the spindle equator, while spindle poles segregate rapidly elongating the spindle. Direct biophysical measurements of forces driving these movements are lacking, but the forces could be learned from the rapid pole and chromosome movements. We analyzed 3D trajectories of chromosomes and spindle poles in the wild-type and PRC1-depleted cells during early prometaphase and reconstructed the spindle forces. This analysis posits that (i) chromosomes do not interact with each other, except for a short-range steric repulsion, (ii) effective pole-chromosome forces are repulsive at short and attractive at long distances, (iii) dominant forces of spindle poles segregation are interpolar in origin. The important implication of these results is that the pole-pole segregation is robust, largely insensitive to regional variations inside the cell and to complex process of chromosome congression. SIGNIFICANCE: Mechanics of prometaphase is crucial for mitotic spindle assembly, yet measurements of forces in prometaphase are lacking. We use movements of centrosomes and chromosomes in prometaphase to reconstruct the spindle forces. We find that the forces are hierarchical, with the interpolar force balance dominating smaller forces between the spindle poles and chromosomes. The important implication of these results is that the pole-pole segregation is robust, largely insensitive to regional variations inside the cell and to complex process of chromosome congression.

The mechanisms underlying both the establishment of mirror (reflection) symmetry and deviations from it in the development of bilateral multicellular organisms remain insufficiently understood. Actin cytoskeletons of individual cells exhibit intrinsic chirality, and a strong correlation exists between single-cell actin fibres’ chiral organisation and the collective alignment of cells confined to rectangular adhesive islands (2D-microtissues). Here, we demonstrate how multicellular chiral patterns can be inferred from the chiral behaviour of actin fibres in individual cells. By analysing chiral actin systems in cells with elliptical and semicircular shapes, representing inner and boundary positions within 2D-microtissues, we defined the rules of chiral motile behaviour and formulated two models of cell alignment: (i) chiral rotation of inner cells and (ii) chiral tilting of boundary cells relative to island edges. In both models, neighbouring cells are mutually aligned. Systematic variation of island area and aspect ratio, combined with dynamic observations, revealed the primary role of boundary cells. Chiral order first emerged at tissue boundaries and then propagated inward. This outside-in mechanism also explains how intrinsically chiral cells can build mirror-symmetric tissues in bilateral organisms: either by reversing cell chirality in one half or by enlarging the tissue to minimise boundary influence.

Mesenchymal cell migration starts with the protrusion of the cell’s leading edge, enabled by the branching growth of the actin network. Two crucial molecular players in protrusion are actin filaments and the Arp2/3 protein complex, the branching agent. Traditionally, protrusion models are intuited from several perturbative experiments. Recent multiplex microscopy imaging data promise to drastically change this paradigm by providing measurements of naturally fluctuating densities of key proteins at the leading edge of unperturbed cells. We report the first attempt to reconstruct the mechanistic protrusion model from such data. We analyze fluctuations of F-actin and Arp2/3 densities and cell edge velocity using phase space and regression analysis to reconstruct linearized stochastic actin-Arp2/3-velocity coupled dynamics. We then build a nonlinear partial differential equation model of these dynamics based on previous knowledge of this system and parsimony. The resulting model recovers the rates of reactions and mechanical and transport processes in the lamellipodium from a single non-perturbative experiment. The model posits that the only essential nonlinearities in the lamellipodial dynamics stem from the retrograde flow of F-actin. The model suggests that the protrusion is dominated by a damped oscillatory cycle resulting from a combination of positive feedback from Arp2/3 to protrusion velocity and negative feedback from F-actin to the velocity. Significance statement: Traditionally, models of intracellular mechanochemistry are intuited from perturbative experiments. Multiplex microscopy imaging changes this by providing measurements of naturally fluctuating densities of key proteins in unperturbed cells. We use such data to reconstruct a mechanistic model of cell leading edge. We build equations for actin system and edge velocity using data, previous knowledge and parsimony. The resulting model recovers the rates of reactions and mechanical and transport processes at the leading edge. The model suggests that the protrusion is dominated by a damped oscillatory cycle resulting from a combination of positive feedback from Arp2/3 to protrusion velocity and negative feedback from F-actin to the velocity.

Living systems transform their shapes via reversible formation of macromolecular structural complexes, leading to deformations at localized sites. Here we report DNA–inorganic flower-shaped crystals with inscribed deformation modes that enable flowers to shrink and bend reversibly. Template-independent DNA polymerization of pH-responsive and inert blocks tune the hierarchical assembly and spatial localization of DNA within flowers. Experiments and simulations demonstrate that reversible, pH-triggered folding of intraflower DNA strands drives reconfiguration of flowers. By contrast, the subflower localization of these contractile DNA motifs dictates the mode of shape change. As microscale flowers close and open, their nanoscale crystal organization changes reversibly, suggesting that mechanical metamorphosis of flowers is transduced across multiple organizational length scales. The adaptability of flowers to environmental changes activates cascaded biocatalytic reactions and reveals gel-encrypted information. Further variation of the DNA polymer sequence, its subcrystal localization and its reversible folding advances a new class of organic–inorganic shape-shifters.

The mechanisms underlying both the establishment of mirror symmetry and deviations from it in development of bilateral multicellular organisms remain insufficiently understood. Actin cytoskeletons of individual cells exhibit intrinsic chirality, and a strong correlation exists between single-cell actin fibres chiral organisation and the collective alignment of cells confined to rectangular adhesive islands (2D-microtissues). Here, we demonstrate how multicellular chiral patterns can be inferred from the chiral behaviour of actin fibres in individual cells. By analysing chiral actin systems in cells with elliptical and semi-circular shapes, representing inner and boundary positions within 2D-microtissues, we defined the rules of chiral motile behaviour and formulated two models of cell alignment: (i) chiral rotation of inner cells and (ii) chiral tilting of boundary cells relative to island edges. In both models, neighbouring cells are also mutually aligned. Systematic variation of island area and aspect ratio, combined with dynamic observations, revealed the primary role of boundary cells. Chiral order first emerged at tissue boundaries and then propagated inward. This outside-in mechanism also explains why mirror-symmetric cell groups are difficult to achieve with uniformly chiral cells, but can be obtained either by reversing chirality in one subgroup or by enlarging the microtissue to minimise boundary influence.

In studying the transport of inclusions in multiphase systems we are often interested in integrated quantities such as the net force and the net velocity of the inclusions. In the reciprocal theorem the known solution to the first and typically easier boundary value problem is used to compute the integrated quantities, such as the net force, in the second problem without the need to solve that problem. Here, we derive a reciprocal theorem for poro-viscoelastic (or biphasic) materials that are composed of a linear compressible solid phase, permeated by a viscous fluid. As an example, we analytically calculate the time-dependent net force on a rigid sphere in response to point forces applied to the elastic network and the Newtonian fluid phases of the biphasic material. We show that when the point force is applied to the fluid phase, the net force on the sphere evolves over time scales that are independent of the distance between the point force and the sphere; in comparison, when the point force is applied to the elastic phase, the time scale for force development increases quadratically with the distance, in line with the scaling of poroelastic relaxation time. Finally, we formulate and discuss how the reciprocal theorem can be applied to other areas, including (i) calculating the network slip on the sphere's surface, (ii) computing the leading-order effects of nonlinearities in the fluid and network forces and stresses, and (iii) calculating self-propulsion in biphasic systems.

Communication in biological systems typically involves enzymatic reactions that occur within fluids confined between the soft, elastic walls of bio-channels and chambers. Through the inherent transformation of chemical to mechanical energy, the fluids can be driven to flow within the confined domains. Through fluid-structure interactions, the confining walls in turn are deformed by and affect this fluid flow. Imbuing synthetic materials with analogous feedback among chemo-mechanical, hydrodynamic and fluid-structure interactions could enable materials to perform self-driven communication and self-regulation. Herein, we develop computational models to determine how chemo-hydro-mechanical feedback affects interactions in biomimetic arrays of chemically active and passive micro-posts anchored in fluid-filled chambers. Once activated, the enzymatic reactions trigger the latter feedback, which generates a surprising variety of long-range, cooperative motion, including self-oscillations and non-reciprocal interactions, which are vital for propagating coherent, directional signals over net distances in fluids. In particular, the array propagates a distinct message; each post interprets the message; and the system responds with a specific mode of organized, collective behavior. This level of autonomous, remote control is relatively rare in synthetic systems, particularly as this system operates without external electronics or power sources and only requires the addition of chemical reactants to function.

Septins are a class of cytoskeletal proteins that preferentially bind to domains of micron-scale curvature on the cell membrane. Studies have shown that amphipathic helix (AH) domains in septin oligomers are essential for septin curvature sensing. Yet, the underlying mechanochemical interactions that modulate this curvature sensing remain ambiguous. Here we use all-atom molecular dynamics alongside a metadynamics enhanced sampling approach to bridge the gap between time and length scales required to optimize and validate experimental design of amphipathic helices. Simulations revealed that the local charge on the termini of an 18-amino-acid AH peptide impacts its helical content and positioning within lipid membranes. These computational observations are confirmed with experiments measuring the binding of synthetic AH constructs with variable helical content and charged termini to lipid vesicles. Taken together, these results identify the helical content of amphipathic helices as a regulator of septin binding affinity to lipid membranes. Additionally, we examined an extended AH sequence including 8 amino acids upstream and downstream of the minimal 18-amino-acid-long AH domain to more closely mimic the native protein in simulations and experiments. Simulations and experiments show that the extended peptide sequence adopts a strong alpha-helical conformation when free in solution, giving rise to a higher affinity to lipid membranes than that of the shorter AH sequence. Together, these results provide insight into how the native septin proteins interact with membranes, and establish general design principles that can guide the interaction of future synthetic materials with lipid membranes in a programmable manner. STATEMENT OF SIGNIFICANCE Understanding how cells sense and react to their shape is necessary for numerous biological processes. Here we explore the interactions between amphipathic helices, a curvature sensing protein motif, and lipid membranes. Using molecular dynamics simulations, enhanced simulation sampling techniques, and experiments, we find that increasing the helical content of the amphipathic helix or adding charged capping sequences yields higher membrane binding affinity. Understanding these parameters for membrane-binding could enable us to interface and regulate native protein functions, as well as guide the design of synthetic curvature-sensing materials that can interact with and deform lipid membranes.

Many of the cell membrane's vital functions are achieved by the self-organization of the proteins and biopolymers embedded in it. The protein dynamics is in part determined by its drag. A large number of these proteins can polymerize to form filaments. In vitro studies of protein–membrane interactions often involve using rigid beads coated with lipid bilayers, as a model for the cell membrane. Motivated by this, we use slender-body theory to compute the translational and rotational resistance of a single filamentous protein embedded in the outer layer of a supported bilayer membrane and surrounded on the exterior by a Newtonian fluid. We first consider the regime where the two layers are strongly coupled through their inter-leaflet friction. We find that the drag along the parallel direction grows linearly with the filament's length and quadratically with the length for the perpendicular and rotational drag coefficients. These findings are explained using scaling arguments and by analysing the velocity fields around the moving filament. We then present and discuss the qualitative differences between the drag of a filament moving in a freely suspended bilayer and a supported membrane as a function of the membrane's inter-leaflet friction. Finally, we briefly discuss how these findings can be used in experiments to determine membrane rheology. In summary, we present a formulation that allows computation of the effects of membrane properties (its curvature, viscosity and inter-leaflet friction), and the exterior and interior three-dimensional fluids’ depth and viscosity on the drag of a rod-like/filamentous protein, all in a unified theoretical framework.