Project description:KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7-3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.
Project description:In this paper, three multiscale methods for coupling of mesoscopic (compartment-based) and microscopic (molecular-based) stochastic reaction-diffusion simulations are investigated. Two of the three methods that will be discussed in detail have been previously reported in the literature; the two-regime method (TRM) and the compartment-placement method (CPM). The third method that is introduced and analysed in this paper is called the ghost cell method (GCM), since it works by constructing a "ghost cell" in which molecules can disappear and jump into the compartment-based simulation. Presented is a comparison of sources of error. The convergent properties of this error are studied as the time step ?t (for updating the molecular-based part of the model) approaches zero. It is found that the error behaviour depends on another fundamental computational parameter h, the compartment size in the mesoscopic part of the model. Two important limiting cases, which appear in applications, are considered: (i) ?t ? 0 and h is fixed; (ii) ?t ? 0 and h ? 0 such that ??t/h is fixed. The error for previously developed approaches (the TRM and CPM) converges to zero only in the limiting case (ii), but not in case (i). It is shown that the error of the GCM converges in the limiting case (i). Thus the GCM is superior to previous coupling techniques if the mesoscopic description is much coarser than the microscopic part of the model.
Project description:Rational synthesis of materials is a long-term challenging issue due to the poor understanding on the formation mechanism of material structure and the limited capability in controlling nanoscale crystallization. The emergent in situ electron microscope provides an insight to this issue. By employing an in situ scanning electron microscope, silver crystallization is investigated in real time, in which a reversible crystallization is observed. To disclose this reversible crystallization, the radicals generated by the irradiation of electron beam are calculated. It is found that the concentrations of radicals are spatiotemporally variable in the liquid cell due to the diffusion and reaction of radicals. The fluctuation of the reductive hydrated electrons and the oxidative hydroxyl radicals in the cell leads to the alternative dominance of the reduction and oxidation reactions. The reduction leads to the growth of silver crystals while the oxidation leads to their dissolution, which results in the reversible silver crystallization. A regulation of radical distribution by electron dose rates leads to the formation of diverse silver structures, confirming the dominant role of local chemical concentration in the structure evolution of materials.
Project description:We report the unexpected discovery of a tandem active template CuAAC-rearrangement process, in which N2 is extruded on the way to the 1,2,3-triazole product to give instead acrylamide rotaxanes. Mechanistic investigations suggest this process is dictated by the mechanical bond, which stabilizes the CuI -triazolide intermediate of the CuAAC reaction and diverts it down the rearrangement pathway; when no mechanical bond is formed, the CuAAC product is isolated.
Project description:Stem cells in organoids self-organize into tissue patterns with unknown mechanisms. Here, we use skin organoids to analyze this process. Cell behavior videos show that the morphological transformation from multiple spheroidal units with morphogenesis competence (CMU) to planar skin is characterized by two abrupt cell motility-increasing events before calming down. The self-organizing processes are controlled by a morphogenetic module composed of molecular sensors, modulators, and executers. Increasing dermal stiffness provides the initial driving force (driver) which activates Yap1 (sensor) in epidermal cysts. Notch signaling (modulator 1) in epidermal cyst tunes the threshold of Yap1 activation. Activated Yap1 induces Wnts and MMPs (epidermal executers) in basal cells to facilitate cellular flows, allowing epidermal cells to protrude out from the CMU. Dermal cell-expressed Rock (dermal executer) generates a stiff force bridge between two CMU and accelerates tissue mixing via activating Laminin and β1-integrin. Thus, this self-organizing coalescence process is controlled by a mechano-chemical circuit. Beyond skin, self-organization in organoids may use similar mechano-chemical circuit structures.
Project description:During cell motion on a substratum, eukaryotic cells project sheetlike lamellipodia which contain a dynamically remodeling three-dimensional actin mesh. A number of regulatory proteins and subtle mechano-chemical couplings determine the lamellipodial protrusion dynamics. To study these processes, we constructed a microscopic physico-chemical computational model, which incorporates a number of fundamental reaction and diffusion processes, treated in a fully stochastic manner. Our work sheds light on the way lamellipodial protrusion dynamics is affected by the concentrations of actin and actin-binding proteins. In particular, we found that protrusion speed saturates at very high actin concentrations, where filament nucleation does not keep up with protrusion. This results in sparse filamentous networks, and, consequently, high resistance forces on individual filaments. We also observed maxima in lamellipodial growth rates as a function of Arp2/3, a nucleating protein, and capping proteins. We provide detailed physical explanations behind these effects. In particular, our work supports the actin-funneling-hypothesis explanation of protrusion speed enhancement at low capping protein concentrations. Our computational results are in agreement with a number of related experiments. Overall, our work emphasizes that elongation and nucleation processes work highly cooperatively in determining the optimal protrusion speed for the actin mesh in lamellipodia.
Project description:Carbon nanotubes can be chemically modified by attaching various functionalities to their surfaces, although harsh chemical treatments can lead to their break-up into graphene nanostructures. On the other hand, direct coupling between functionalities bound on individual nanotubes could lead to, as yet unexplored, spontaneous chemical reactions. Here we report an ambient mechano-chemical reaction between two varieties of nanotubes, carrying predominantly carboxyl and hydroxyl functionalities, respectively, facilitated by simple mechanical grinding of the reactants. The purely solid-state reaction between the chemically differentiated nanotube species produces condensation products and unzipping of nanotubes due to local energy release, as confirmed by spectroscopic measurements, thermal analysis and molecular dynamic simulations.
Project description:Reaction-diffusion systems are used to represent many biological and physical phenomena. They model the random motion of particles (diffusion) and interactions between them (reactions). Such systems can be modelled at multiple scales with varying degrees of accuracy and computational efficiency. When representing genuinely multiscale phenomena, fine-scale models can be prohibitively expensive, whereas coarser models, although cheaper, often lack sufficient detail to accurately represent the phenomenon at hand. Spatial hybrid methods couple two or more of these representations in order to improve efficiency without compromising accuracy. In this paper, we present a novel spatial hybrid method, which we call the auxiliary region method (ARM), which couples PDE- and Brownian-based representations of reaction-diffusion systems. Numerical PDE solutions on one side of an interface are coupled to Brownian-based dynamics on the other side using compartment-based 'auxiliary regions'. We demonstrate that the hybrid method is able to simulate reaction-diffusion dynamics for a number of different test problems with high accuracy. Furthermore, we undertake error analysis on the ARM which demonstrates that it is robust to changes in the free parameters in the model, where previous coupling algorithms are not. In particular, we envisage that the method will be applicable for a wide range of spatial multi-scales problems including filopodial dynamics, intracellular signalling, embryogenesis and travelling wave phenomena.
Project description:Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca(2+) oscillations that triggered increases in the amplitude of mitochondrial Ca(2+) oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca(2+) oscillations and of mitochondrial electron transport indicated that the ROS production was Ca(2+) dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca(2+) oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lung's proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs.
Project description:The aim of this work is to quantify the spatio-temporal dynamics of flow-driven amoeboid locomotion in small (~100 µm) fragments of the true slime mold Physarum polycephalum. In this model organism, cellular contraction drives intracellular flows, and these flows transport the chemical signals that regulate contraction in the first place. As a consequence of these non-linear interactions, a diversity of migratory behaviors can be observed in migrating Physarum fragments. To study these dynamics, we measure the spatio-temporal distributions of the velocities of the endoplasm and ectoplasm of each migrating fragment, the traction stresses it generates on the substratum, and the concentration of free intracellular calcium. Using these unprecedented experimental data, we classify migrating Physarum fragments according to their dynamics, finding that they often exhibit spontaneously coordinated waves of flow, contractility and chemical signaling. We show that Physarum fragments exhibiting symmetric spatio-temporal patterns of endoplasmic flow migrate significantly slower than fragments with asymmetric patterns. In addition, our joint measurements of ectoplasm velocity and traction stress at the substratum suggest that forward motion of the ectoplasm is enabled by a succession of stick-slip transitions, which we conjecture are also organized in the form of waves. Combining our experiments with a simplified convection-diffusion model, we show that the convective transport of calcium ions may be key for establishing and maintaining the spatiotemporal patterns of calcium concentration that regulate the generation of contractile forces.