Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10–20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed

Extrapolation-CAM Theory for Critical Exponents

By intentionally underestimating the rate of convergence of exact-diagonalization values for the mass or energy gaps of finite systems, we form families of sequences of gap estimates. The gap estimates cross zero with generically nonzero linear terms in their Taylor expansions, so that $\nu = 1$ for each member of these sequences of estimates. Thus, the Coherent Anomaly Method can be used to determine $\nu$. Our freedom in deciding exactly how to underestimate the convergence allows us to choose the sequence that displays the clearest coherent anomaly. We demonstrate this approach on the two-dimensional ferromagnetic Ising model, for which $\nu = 1$. We also use it on the three-dimensional ferromagnetic Ising model, finding $\nu \approx 0.629$, in good agreement with other estimates.Comment: 21 pages, Submitted to Journal of Physics A; new section added discussing rate of convergence and relation to Finite-Size Scalin

Molecular Chemical Engines: Pseudo-Static Processes and the Mechanism of Energy Transduction

We propose a simple theoretical model for a molecular chemical engine that catalyzes a chemical reaction and converts the free energy released by the reaction into mechanical work. Binding and unbinding processes of reactant and product molecules to and from the engine are explicitly taken into account. The work delivered by the engine is calculated analytically for infinitely slow (``pseudo-static'') processes, which can be reversible (quasi-static) or irreversible, controlled by an external agent. It is shown that the work larger than the maximum value limited by the second law of thermodynamics can be obtained in a single cycle of operation by chance, although the statistical average of the work never exceeds this limit and the maximum work is delivered if the process is reversible. The mechanism of the energy transductionis also discussed.Comment: 8 pages, 3 figues, submitted to J. Phys. Soc. Jp

Catalysis and rotation of F-1 motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40 degrees substep rotation

F-1, a water-soluble portion of FoF1-ATP synthase, is an ATIP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 801 substep rotation, interim dwell, and rapid 40degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATIP binding, correspond to these events. To discover which steps, we analyzed rotations of F-1 subcomplex (alpha(3)beta(3)gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATIP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F, and hydrolysis of a slowly hydrolyzable substrate ATPgammaS (adenosine 5'-[gamma-thio]triphosphate) by wild-type F-1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPgammaS hydrolysis by the mutant F-1 ensure that cleavage of ATIP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F-1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell

Efficiency of Energy Transduction in a Molecular Chemical Engine

A simple model of the two-state ratchet type is proposed for molecular chemical engines that convert chemical free energy into mechanical work and vice versa. The engine works by catalyzing a chemical reaction and turning a rotor. Analytical expressions are obtained for the dependences of rotation and reaction rates on the concentrations of reactant and product molecules, from which the performance of the engine is analyzed. In particular, the efficiency of energy transduction is discussed in some detail.Comment: 4 pages, 4 fugures; title modified, figures 2 and 3 modified, content changed (pages 1 and 4, mainly), references adde

Direct Observation of the Myosin Va Recovery Stroke That Contributes to Unidirectional Stepping along Actin

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed “strokes”; the “power stroke” is the force-generating swinging of the myosin light chain–binding “neck” domain relative to the motor domain “head” while bound to actin; the “recovery stroke” is the necessary initial motion that primes, or “cocks,” myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a “hand over hand” mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥5 kBT of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va

Bose-Einstein condensation in multilayers

The critical BEC temperature $T_{c}$ of a non interacting boson gas in a layered structure like those of cuprate superconductors is shown to have a minimum $T_{c,m}$, at a characteristic separation between planes $a_{m}$. It is shown that for $a<a_{m}$, $T_{c}$ increases monotonically back up to the ideal Bose gas $T_{0}$ suggesting that a reduction in the separation between planes, as happens when one increases the pressure in a cuprate, leads to an increase in the critical temperature. For finite plane separation and penetrability the specific heat as a function of temperature shows two novel crests connected by a ridge in addition to the well-known BEC peak at $T_{c}$ associated with the 3D behavior of the gas. For completely impenetrable planes the model reduces to many disconnected infinite slabs for which just one hump survives becoming a peak only when the slab widths are infinite.Comment: Four pages, four figure

Direct Observation of Strand Passage by DNA-Topoisomerase and Its Limited Processivity

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51±0.33 µm (11±6 turns) of a braid was unlinked in a burst of reactions taking 8±4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25–37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ∼100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ∼10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ∼10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments

Diffusion-Weighted MRI for Verification of Electroporation-Based Treatments

Clinical electroporation (EP) is a rapidly advancing treatment modality that uses electric pulses to introduce drugs or genes into, e.g., cancer cells. The indication of successful EP is an instant plasma membrane permeabilization in the treated tissue. A noninvasive means of monitoring such a tissue reaction represents a great clinical benefit since, in case of target miss, retreatment can be performed immediately. We propose diffusion-weighted magnetic resonance imaging (DW-MRI) as a method to monitor EP tissue, using the concept of the apparent diffusion coefficient (ADC). We hypothesize that the plasma membrane permeabilization induced by EP changes the ADC, suggesting that DW-MRI constitutes a noninvasive and quick means of EP verification. In this study we performed in vivo EP in rat brains, followed by DW-MRI using a clinical MRI scanner. We found a pulse amplitude–dependent increase in the ADC following EP, indicating that (1) DW-MRI is sensitive to the EP-induced changes and (2) the observed changes in ADC are indeed due to the applied electric field