A proposed time-resolved X-ray scattering approach to track local and global conformational changes in membrane transport proteins

Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed by halorhodopsin as a model system, we combine molecular dynamics simulations with X-ray scattering calculations to demonstrate how small-molecule time-resolved X-ray scattering can be extended to the study of membrane transport processes. In particular, by introducing strongly scattering atoms to label specific positions within the protein and substrate, the technique of time-resolved wide-angle X-ray scattering can reveal both local and global conformational changes. This approach simultaneously enables the direct visualization of global rearrangements and substrate movement, crucial concepts that underpin the alternating access paradigm for membrane transport proteins.     

The relation between local and global transport parameters in an epithelial membrane having an asymmetric transport mechanism

The relationship between local and global transport parameters is derived for an epithelial membrane having series-parallel topology and an asymmetric transport mechanism at its apical and/or basolateral cell membranes. The result shows that the difference in the local forward and backward solute permeability coefficients is manifest in the global volume flow equation as distinct forward and backward reflection coefficients. The analytical result is applicable to the first order (low concentration) domain of Michaelis-Menten kinetics and a procedure is given for simulating the remainder of the domain on SPICE2.     

Raft Formation in Lipid Bilayers Coupled to Curvature

We present computer simulations of a membrane in which the local composition is coupled to the local membrane curvature. At high temperatures (i.e., above the temperature of macroscopic phase separation), finite-sized transient domains are observed, reminiscent of lipid rafts. The domain size is in the range of hundred nanometers, and set by the membrane elastic properties. These findings are in line with the notion of the membrane as a curvature-induced microemulsion. At low temperature, the membrane phase separates. The transition to the phase-separated regime is continuous and belongs to the two-dimensional Ising universality class when the coupling to curvature is weak, but becomes first-order for strong curvature-composition coupling.     

Effect of unstirred layers on binding and reaction kinetics at a membrane surface

The effects of solution unstirred layers on the time course of chemical reactions and transport processes at a membrane surface are determined. A set of equations which describes non-steady-state diffusion through an unstirred layer coupled with chemical reaction at a membrane surface or transport through a membrane is developed. A numerical solution to the equations is obtained by uncoupling diffusive and chemical processes in an iterative manner. The diffusive process is solved by the Crank-Nicolson method; the chemical process is solved by integrating the differential equations describing the kinetics. Diffusive processes in one dimension, in three dimensions, and in the presence of an arbitrary potential near the membrane surface are solved. General characteristics of the calculated reaction time course are discussed using surface binding and membrane transport examples. Small, neglected, unstirred layers are shown to sometimes yield erroneous values of rate parameters for a surface reaction and to simulate competitive reaction kinetics. Experimental approaches for measuring unstirred layer thickness are reviewed.     

Gradual regime shifts in spatially extended ecosystems

Ecosystem regime shifts are regarded as abrupt global transitions from one stable state to an alternative stable state, induced by slow environmental changes or by global disturbances. Spatially extended ecosystems, however, can also respond to local disturbances by the formation of small domains of the alternative state. Such a response can lead to gradual regime shifts involving front propagation and the coalescence of alternative-state domains. When one of the states is spatially patterned, a multitude of intermediate stable states appears, giving rise to step-like gradual shifts with extended pauses at these states. Using a minimal model, we study gradual state transitions and show that they precede abrupt transitions. We propose indicators to probe gradual regime shifts, and suggest that a combination of abrupt-shift indicators and gradual-shift indicators might be needed to unambiguously identify regime shifts. Our results are particularly relevant to desertification in drylands where transitions to bare soil take place from spotted vegetation, and the degradation process appears to involve step-like events of local vegetation mortality caused by repeated droughts.     

Systems approach to the study of drug transport across membranes using suspension cultures of mammalian cells V. Simultaneous passive transport and biosynthesis

A physical model is described for the simultaneous enzymatic bioconversion of a nonelectrolyte solute and the passive transport of both the solute and product of the enzymatic reaction out of cells in culture suspension. The plasma membrane is assumed to be the rate-determining transport barrier. This model provides the basis for the experimental design and analysis of the Michaelis-Menten kinetic parameters of simple enzymatic reactions in situ, the phenomenological transport parameters and other factors. The primary set of differential equations describing the quasisteady state rate of change in the concentration of the solute and product within the cell due to enzyme reaction and transport are given. These are nonlinear and must be solved by numerical methods. However, analytical mathematical expressions have been derived for various cases in the limit when the rate of enzymatic reaction is first or zero order.     

Regime Shifts in the Anthropocene: Drivers,Risks, and Resilience

Many ecosystems can experience regime shifts: surprising, large and persistent changes in the function and structure of ecosystems. Assessing whether continued global change will lead to further regime shifts, or has the potential to trigger cascading regime shifts has been a central question in global change policy. Addressing this issue has, however, been hampered by the focus of regime shift research on specific cases and types of regime shifts. To systematically assess the global risk of regime shifts we conducted a comparative analysis of 25 generic types of regime shifts across marine, terrestrial and polar systems; identifying their drivers, and impacts on ecosystem services. Our results show that the drivers of regime shifts are diverse and co-occur strongly, which suggests that continued global change can be expected to synchronously increase the risk of multiple regime shifts. Furthermore, many regime shift drivers are related to climate change and food production, whose links to the continued expansion of human activities makes them difficult to limit. Because many regime shifts can amplify the drivers of other regime shifts, continued global change can also be expected to increase the risk of cascading regime shifts. Nevertheless, the variety of scales at which regime shift drivers operate provides opportunities for reducing the risk of many types of regime shifts by addressing local or regional drivers, even in the absence of rapid reduction of global drivers.     

Stability properties of elementary dynamic models of membrane transport

Living cells are characterized by their capacity to maintain a stable steady state. For instance, cells are able to conserve their volume, internal ionic composition and electrical potential difference across the plasma membrane within values compatible with the overall cell functions. The dynamics of these cellular variables is described by complex integrated models of membrane transport. Some clues for the understanding of the processes involved in global cellular homeostasis may be obtained by the study of the local stability properties of some partial cellular processes. As an example of this approach, I perform, in this study, the neighborhood stability analysis of some elementary integrated models of membrane transport. In essence, the models describe the rate of change of the intracellular concentration of a ligand subject to active and passive transport across the plasma membrane of an ideal cell. The ligand can be ionic or nonionic, and it can affect the cell volume or the plasma membrane potential. The fundamental finding of this study is that, within the physiological range, the steady states are asymptotically stable. This basic property is a necessary consequence of the general forms of the expressions employed to describe the active and passive fluxes of the transported ligand.     

Effect of membrane flow on the capture of receptors by coated pits. Theoretical results.

Coated pits trap cell surface receptors and mediate their internalization. Once internalized, many receptors recycle back to the cell surface. When recycled receptors are inserted into the plasma membrane, they move until they are again trapped in coated pits. The mechanisms for moving receptors from their insertion sites to coated pits are unknown. Unaided diffusion as the transport mechanism is consistent with the observed kinetics of receptor recycling. Another candidate for the transport mechanism is convection. For receptors that recycle to random positions on the cell surface, or to restricted regions about coated pits, we assess the importance of convective flow in the transport of receptors to coated pits. First we consider local flows set up by the formation of coated pits and their transformation into coated vesicles. As coated pits form and round into coated vesicles, surrounding membrane is drawn inward, creating flows directed toward the coated pit centers. We show that unless the lifetime of a coated pit is very short, 10 s or less, such local flows have a negligible effect on the time it takes receptors to reach coated pits. We also show that they are unlikely to be the mechanism that keeps receptors that have reached coated pits trapped within coated pits until they are internalized. Finally we calculate the mean time tau for a diffusing receptor to reach a coated pit in the presence of membrane flow that is constant in magnitude and direction, as may occur on moving cells. We show that for typical membrane flow velocities, tau can be reduced significantly from its value in the absence of flow. For example, a velocity v = 2.8 micron/min cuts the mean transport time in half.     

The effect of membrane heterogeneity on the predictability of fluxes,with application to the cornea

Many phenomenological treatments of biological membrane transport are based on the assumption that the membrane consists of a single class of passive transport paths; i.e. that the membrane is simple. Simplicity is assumed both in the measurement of the membrane's transport coefficients and in the use of these coefficients to predict membrane fluxes. Such a procedure will in general lead to an error in the prediction of solute flux across parallel arrays. The error depends on the distribution of the reflection coefficients of the parallel paths and upper and lower bounds on it are given in terms of conventionally measured transport coefficients. The frictional representation of electrolyte transport across membranes possessing metabolic pumps is generalized to take this structure effect into account. The flux error resulting from the neglect of membrane heteroreflectivity is essentially the same for nonelectrolytes and electrolytes, irrespective of whether phenomenological or frictional membrane transport properties are used. It is shown that some information about transport structure can be obtained from global measurements made without regard for the organization of pathways across the membrane.The values of the measured transport coefficients of the corneal epithelium and endothelium imply that these cell layers are heteroreflective. Analysis of corneal transport, taking structure effects into account, shows that the corneal thickness may be intrinsically insensitive to tear tonicity, by a mechanism which may be of more general homeostatic significance.     

Range-size,local abundance and germination niche-breadth in Mediterranean plants of two life-forms

The relationships between rarity (i.e., range-size, local abundance) and niche-breadth can be important to assess the risks the species face under global change, namely those resulting from fire regime change. In fire areas, germination is critical for establishing after fire for many species. We examined the relationships between rarity and germination niche-breadth for 53 plant species of two life-forms (chamaephytes and hemicryptophytes) growing in Eastern Spain. Rarity was measured as geographic range-size and as local abundance. Local abundance was evaluated at two sites differing in their post-fire successional status (a recently burned area and a long-unburned one). Germination niche-breadth was measured as the mean germination evenness index from four germination experiments that subjected the seeds to various germinating conditions characteristics of fire environments. Correlations between rarity (range-size, local abundance) and niche-breadth were calculated in cross-species mode or by way of phylogenetically independent contrasts, and either for the 53 species (all-species set) or for each of two life-form groups (chamaephytes, hemicryptophytes). In general, no significant correlations were found between the rarity measures and germination niche-breadth for the all-species set. However, significant correlations emerged when the analyses were done separating species by life-forms. Germination niche-breadth was positively correlated with range-size for chamaephytes, and negatively for hemicryptophytes. In addition, germination niche-breadth was uncorrelated, or negatively so, with local abundance for chamaephytes and hemicryptophytes, respectively. While no correlation between range-size and local abundance was found for chamaephytes, a negative one was obtained for hemicryptophytes. We conclude that rarity/germination-niche relationships varied by life-form. This pattern of relationships was obscured when all species were joined in a single group. Based on the contrasting patterns of correlations obtained for each life-form we argue that the changes in the germinating environment caused by alterations in fire regime are likely to differentially affect these two groups of species.     

An actin-dependent mechanism for long-range vesicle transport

Intracellular transport is vital for the function, survival and architecture of every eukaryotic cell. Long-range transport in animal cells is thought to depend exclusively on microtubule tracks. This study reveals an unexpected actin-dependent but microtubule-independent mechanism for long-range transport of vesicles. Vesicles organize their own actin tracks by recruiting the actin nucleation factors Spire1, Spire2 and Formin-2, which assemble an extensive actin network from the vesicles' surfaces. The network connects the vesicles with one another and with the plasma membrane. Vesicles move directionally along these connections in a myosin-Vb-dependent manner to converge and to reach the cell surface. The overall outward-directed movement of the vesicle-actin network is driven by recruitment of vesicles to the plasma membrane in the periphery of the oocyte. Being organized in a dynamic vesicle-actin network allows vesicles to move in a local random manner and a global directed manner at the same time: they can reach any position in the cytoplasm, but also move directionally to the cell surface as a collective. Thus, collective movement within a network is a powerful and flexible mode of vesicle transport.     

Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides. The coupling between transport and phosphorylation in inside-out vesicles

The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the literature. We have tested the coupling by analysing the kinetics of fructose phosphorylation by inside-out vesicles of Rhodopseudomonas sphaeroides. We conclude that fructose indeed has to enter the vesicle before it can be phosphorylated and therefore cannot be phosphorylated from the cytoplasmic side of the membrane. The Km of the phosphorylation reaction is 8 microM. The diffusion of fructose into the vesicle is a reaction that is also catalysed by the components of the phosphotransferase system. The undirectional flux from the cytoplasmic side of the membrane to the periplasmic side is a slow process with a Km of 4 mM and is rate-limiting over a large external fructose concentration range. In summary there is no phosphorylation without transport, but there is transport without phosphorylation.     

Changes of nuclear membrane lipid composition affect RNA nucleocytoplasmic transport.

We have previously shown that the nuclear membrane fluidity of rat liver, measured by fluorescence anisotropy of two probes, is higher in the hydrophobic core, with respect to the bilayer surface, in newborn rats compared to adult rats. The aim of the present research is to investigate whether the nuclear membrane fluidity influences RNA nucleocytoplasmic transport. To this end two experimental models were used: the fluidity of nuclear membrane isolated from adult rats was increased by a choline base exchange reaction, which is known to be accompanied by an increase of phosphatidylcholine unsaturated fatty acids, whereas that of nuclear membrane isolated from newborn rats was decreased by incubation with dimyristoylphosphatidylcholine-cholesterol liposomes. The RNA efflux, evaluated by using [3H]uridine, significantly increased in the adult nuclear membrane submitted to choline base exchange reaction, whereas a strong decrease in the newborn nuclear membrane enriched with cholesterol was found. The activity of nucleoside triphosphatase, a nuclear membrane-associated enzyme which is correlated with mRNA transport, showed parallel variations. Therefore, for the first time, we have provided evidence that the nuclear membrane fluidity plays a regulatory role in RNA nucleocytoplasmic transport, although the mechanism by which this effect takes place remains to be clarified.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Mitochondrial protein biogenesis: The essential functions of chaperones and their cofactors during preprotein translocation

Most mitochondrial proteins have to be imported from the cytosol through both mitochondrial membranes to their final localization. A dedicated translocation machinery is responsible for the specific recognition and the membrane transport of mitochondrial precursor proteins. Protein translocase complexes integrated into both mitochondrial membranes cooperate closely with receptor proteins at the surface and provide aqueous transport channels through the membranes. Energy for the membrane insertion is provided by the electric potential across the mitochondrial inner membrane. However, full translocation of the polypeptide chain requires ATP hydrolysis in the matrix. The responsible ATPase enzyme is a member of an ubiquitous family of molecular chaperones, the mitochondrial heat shock protein of 70 kDa (mtHsp70). A physical and functional interaction with a set of cofactors is indispensable for the translocation function of mtHsp70. By a specific and nucleotide-dependent binding to the inner membrane translocase component Tim44, the soluble chaperone mtHsp70 is anchored directly at the site of preprotein membrane insertion. The nucleotide exchange factor Mge1 enhances the ATPase activity of mtHsp70 and is required for the preprotein import reaction. Two novel proteins, Pam18 and Pam16, members of the inner membrane translocation channel, are required to couple the ATPase activity of mtHsp70 to the preprotein import reaction. We have collected experimental evidence indicating that mtHsp70 generates an inward directed translocation force on the polypeptide chain in transit by an ATP-regulated direct interaction with the precursor protein. The force generation results in the movement and active unfolding of the preprotein domains during the translocation process. Taken together, the chaperone mtHsp70 with its accessory proteine forms an import motor complex for mitochondrial preproteins that is driven by the hydrolysis of ATP.     

Three-dimensional numerical approach to investigate the substrate transport and conversion in an immobilized enzyme reactor

This numerical study evaluates the momentum and mass transfer in an immobilized enzyme reactor. The simulation is based on the solution of the three-dimensional Navier-Stokes equation and a scalar transport equation with a sink term for the transport and the conversion of substrate to product. The reactor consists of a container filled with 20 spherical enzyme carriers. Each of these carriers is covered with an active enzyme layer where the conversion takes place. To account for the biochemical activity, the sink term in the scalar transport equation is represented by a standard Michaelis-Menten approach. The simulation gives detailed information of the local substrate and product concentrations with respect to external and internal transport limitations. A major focus is set on the influence of the substrate transport velocity on the catalytic process. For reactor performance analysis the overall and the local transport processes are described by a complete set of dimensionless variables. The interaction between substrate concentration, velocity, and efficiency of the process can be studied with the help of these variables. The effect of different substrate inflow concentrations on the process can be seen in relation to velocity variations. The flow field characterization of the system makes it possible to understand fluid mechanical properties and its importance to transport processes. The distribution of fluid motion through the void volume has different properties in different parts of the reactor. This phenomenon has strong effects on the arrangement of significantly different mass transport areas as well as on process effectiveness. With the given data it is also possible to detect zones of high, low, and latent enzymatic activity and to determine whether the conversion is limited due to mass transfer or reaction resistances.     

Rigidification of neutral lipid bilayers in the presence of salts

We studied the influence of sodium and calcium chloride on the global and local membrane properties of fluid palmitoyl-oleoyl phosphatidylcholine bilayers, applying synchrotron small-angle x-ray diffraction, spin-labeling electron paramagnetic resonance spectroscopy, and differential scanning calorimetry, as well as simultaneous density and acoustic measurements. The salt concentration was varied over a wide range from 0 to 5 M. We found that NaCl leads to a continuous swelling of the bilayers, whereas the behavior of the bilayer separation dW in the presence of CaCl2 is more complex, showing an initial large dW value, which decreased upon further addition of salt and finally increased again in the high concentration regime. This can be understood by a change of balance between electrostatic and van der Waals interactions. We were further able to show that both salts lead to a significant increase of order within the lipid bilayer, leading to a decrease of bilayer elasticity and shift of main phase transition temperature. This effect is more pronounced for Ca2+, and occurs mainly in the high salt-concentration regime. Thus, we were able to reconcile previous controversies between molecular dynamics simulations and x-ray diffraction experiments regarding the effect of salts on neutral lipid bilayers.