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Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs’ ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.

A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell’s interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM’s architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay’s potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM’s mesh size (correlation length) to range from ∼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.

In vertebrate eyes, the rod photoreceptor has a modified cilium with an extended cylindrical structure specialized for phototransduction called the outer segment (OS). The OS has numerous stacked membrane disks and can bend or break when subjected to mechanical forces. The OS exhibits axial structural variation, with extended bands composed of a few hundred membrane disks whose thickness is diurnally modulated. Using high-resolution confocal microscopy, we have observed OS flexing and disruption in live transgenic Xenopus rods. Based on the experimental observations, we introduce a coarse-grained model of OS mechanical rigidity using elasticity theory, representing the axial OS banding explicitly via a spring-bead model. We calculate a bending stiffness of ∼105 nN⋅μm2, which is seven orders-of-magnitude larger than that of typical cilia and flagella. This bending stiffness has a quadratic relation to OS radius, so that thinner OS have lower fragility. Furthermore, we find that increasing the spatial frequency of axial OS banding decreases OS rigidity, reducing its fragility. Moreover, the model predicts a tendency for OS to break in bands with higher spring number density, analogous to the experimental observation that transgenic rods tended to break preferentially in bands of high fluorescence. We discuss how pathological alterations of disk membrane properties by mutant proteins may lead to increased OS rigidity and thus increased breakage, ultimately contributing to retinal degeneration.

Although progress has been made to determine the native fold of a polypeptide from its primary structure, the diversity of pathways that connect the unfolded and folded states has not been adequately explored. Theoretical and computational studies predict that proteins fold through parallel pathways on funneled energy landscapes, although experimental detection of pathway diversity has been challenging. Here, we exploit the high translational symmetry and the direct length variation afforded by linear repeat proteins to directly detect folding through parallel pathways. By comparing folding rates of consensus ankyrin repeat proteins (CARPs), we find a clear increase in folding rates with increasing size and repeat number, although the size of the transition states (estimated from denaturant sensitivity) remains unchanged. The increase in folding rate with chain length, as opposed to a decrease expected from typical models for globular proteins, is a clear demonstration of parallel pathways. This conclusion is not dependent on extensive curve-fitting or structural perturbation of protein structure. By globally fitting a simple parallel-Ising pathway model, we have directly measured nucleation and propagation rates in protein folding, and have quantified the fluxes along each path, providing a detailed energy landscape for folding. This finding of parallel pathways differs from results from kinetic studies of repeat-proteins composed of sequence-variable repeats, where modest repeat-to-repeat energy variation coalesces folding into a single, dominant channel. Thus, for globular proteins, which have much higher variation in local structure and topology, parallel pathways are expected to be the exception rather than the rule.

Because fibroblasts deposit the collagen matrix that determines the mechanical integrity of scar tissue, altering fibroblast invasion could alter wound healing outcomes. Anisotropic mechanical boundary conditions (restraint, stretch, or tension) could affect the rate of fibroblast invasion, but their importance relative to the prototypical drivers of fibroblast infiltration during wound healing—cell and chemokine concentration gradients—is unknown. We tested whether anisotropic mechanical boundary conditions affected the directionality and speed of fibroblasts migrating into a three-dimensional model wound, which could simultaneously expose fibroblasts to mechanical, structural, steric, and chemical guidance cues. We created fibrin-filled slits in fibroblast-populated collagen gels and applied uniaxial mechanical restraint along the short or long axis of the fibrin wounds. Anisotropic mechanical conditions increased the efficiency of fibroblast invasion by guiding fibroblasts without increasing their migration speed. The migration behavior could be modeled as a biased random walk, where the bias due to multiple guidance cues was accounted for in the shape of a displacement orientation probability distribution. Taken together, modeling and experiments suggested an effect of strain anisotropy, rather than strain-induced fiber alignment, on fibroblast invasion.

The motor head of kinesin carries out microtubule binding, ATP hydrolysis, and force generation. Despite a high level of sequence and structural conservation, subtle variations in subdomains of the motor head determine family-specific properties. In particular, both Kinesin-1 (Kin-1) and Kinesin-5 (Kin-5) walk processively to the microtubule plus-end, yet show distinct motility characteristics suitable for their functions. We studied chimeric Kin-1/Kin-5 constructs with a combination of single molecule motility assays and molecular dynamics simulations to demonstrate that Kin-5 possesses a force-generating element similar to Kin-1, i.e., the cover-neck bundle. Furthermore, the Kin-5 neck linker makes additional contacts with the core of the motor head via loop L13, which putatively compensates for the shorter cover-neck bundle of Kin-5. Our results indicate that Kin-1 is mechanically optimized for individual cargo transport, whereas Kin-5 does not necessarily maximize its mechanical performance. Its biochemical rates and enhanced force sensitivity may instead be beneficial for operation in a group of motors. Such variations in subdomains would be a strategy for achieving diversity in motility with the conserved motor head.

We report a model describing the various stages of dorsal closure of Drosophila. Inspired by experimental observations, we represent the amnioserosa by 81 hexagonal cells that are coupled mechanically through the position of the nodes and the elastic forces on the edges. In addition, each cell has radial spokes representing actin filaments on which myosin motors can attach and exert contractile forces on the nodes, the attachment being controlled by a signaling molecule. Thus, the model couples dissipative cell and tissue motion with kinetic equations describing the myosin and signal dynamics. In the early phase, amnioserosa cells oscillate as a result of coupling among the chemical signaling, myosin attachment/detachment, and mechanical deformation of neighboring cells. In the slow phase, we test two ratcheting mechanisms suggested by experiments: an internal ratchet by the apical and junctional myosin condensates, and an external one by the supracellular actin cables encircling the amnioserosa. Within the range of parameters tested, the model predictions suggest the former as the main contributor to cell and tissue area reduction in this stage. In the fast phase of dorsal closure, cell pulsation is arrested, and the cell and tissue areas contract consistently. This is realized in the model by gradually shrinking the resting length of the spokes. Overall, the model captures the key features of dorsal closure through the three distinct phases, and its predictions are in good agreement with observations.

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-μm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.

Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed “catastrophes”, affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system’s “aging”, gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies.

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKII delta) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKII delta and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (similar to 30%) passive force reduction caused by CaMKII delta and a much smaller (similar to 6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKII delta can alter the passive force of myocytes in the heart (although CaMKII delta appears to be more potent) during physiological and pathophysiological states

The charge transporting activity of the Na+,K+-ATPase depends on its surrounding electric field. To isolate which steps of the enzyme’s reaction cycle involve charge movement we have investigated the response of the voltage-sensitive fluorescent probe RH421 to interaction of the protein with benzyltriethylammonium (BTEA), which binds from the extracellular medium to the Na+,K+-ATPase’s transport sites in competition with Na+ and K+, but isn’t occluded within the protein. We find that only the occludable ions Na+, K+, Rb+ and Cs+ cause a drop in RH421 fluorescence. We conclude that RH421 detects intramembrane electric field strength changes arising from charge transport associated with conformational changes occluding the transported ions within the protein, not the electric fields of the bound ions themselves. This appears at first to conflict with electrophysiological studies suggesting extracellular Na+ or K+ binding in a high field access channel is a major electrogenic reaction of the Na+,K+-ATPase. All results can be explained consistently if ion occlusion involves local deformations in the lipid membrane surrounding the protein occurring simultaneously with conformational changes necessary for ion occlusion. The most likely origin of the RH421 fluorescence response is a change in membrane dipole potential caused by membrane deformation. Australian Research Council, National Health and Medical Research Council (Australia)

Cytokinesis is the process of physical cleavage at the end of cell division; it proceeds by ingression of an acto-myosin furrow at the equator of the cell. Its failure leads to multinucleated cells and is a possible cause of tumorigenesis. Here, we calculate the full dynamics of furrow ingression and predict cytokinesis completion above a well-defined threshold of equatorial contractility. The cortical acto-myosin is identified as the main source of mechanical dissipation and active forces. Thereupon, we propose a viscous active nonlinear membrane theory of the cortex that explicitly includes actin turnover and where the active RhoA signal leads to an equatorial band of myosin overactivity. The resulting cortex deformation is calculated numerically, and reproduces well the features of cytokinesis such as cell shape and cortical flows toward the equator. Our theory gives a physical explanation of the independence of cytokinesis duration on cell size in embryos. It also predicts a critical role of turnover on the rate and success of furrow constriction. Scaling arguments allow for a simple interpretation of the numerical results and unveil the key mechanism that generates the threshold for cytokinesis completion: cytoplasmic incompressibility results in a competition between the furrow line tension and the cell poles’ surface tension.

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na(+) ions and the countertransport of a K(+) ion. In this study, we use an EAAT3 homology model to calculate the pKa of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pKa of this residue and favors the release of the proton to solution. Removing the remaining Na(+) ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pKa of E374 with a K(+) ion bound in three possible sites, we show that binding of the K(+) ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K(+) ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.

In the last decade it has become evident that disordered states of proteins play important physiological and pathological roles and that the transient tertiary interactions often present in these systems can play a role in their biological activity. The structural characterization of such states has so far largely relied on ensemble representations, which in principle account for both their local and global structural features. However, these approaches are inherently of low resolution due to the large number of degrees of freedom of conformational ensembles and to the sparse nature of the experimental data used to determine them. Here, we overcome these limitations by showing that tertiary interactions in disordered states can be mapped at high resolution by fitting paramagnetic relaxation enhancement data to a small number of conformations, which can be as low as one. This result opens up the possibility of determining the topology of cooperatively collapsed and hidden folded states when these are present in the vast conformational landscape accessible to disordered states of proteins. As a first application, we study the long-range tertiary interactions of acid-unfolded apomyoglobin from experimentally measured paramagnetic relaxation enhancement data.

A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover.

Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (≤0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s−1, similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network.

Computational simulations using a two-dimensional lattice-Boltzmann immersed boundary method were conducted to investigate the motion of platelets near a vessel wall and close to an intravascular thrombus. Physiological volume fractions of deformable red blood cells and rigid platelet-size elliptic particles were studied under arteriolar flow conditions. Tumbling of platelets in the red-blood-cell depleted zone near the vessel walls was strongly influenced by nearby red blood cells. The thickness of the red-blood-cell depleted zone was greatly reduced near a thrombus, and platelets in this zone were pushed close to the surface of the thrombus to distances that would facilitate their cohesion to it. The distance, nature, and duration of close platelet-thrombus encounters were influenced by the porosity of the thrombus. The strong influence on platelet-thrombus encounters of red-blood-cell motion and thrombus porosity must be taken into account to understand the dynamics of platelet attachment to a growing thrombus.

Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases.

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation.

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca(2+) spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca(2+), Mg(2+), and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca(2+) sparks, Ca(2+) blinks, and Ca(2+) spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca(2+) dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca(2+) dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca(2+), as well as the time course of local Ca(2+) gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca(2+) spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca(2+) spark initiation and subsequent Ca(2+) spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca(2+) in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca(2+) spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca(2+) flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca(2+) sensitivities, Ca(2+) spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Animal life without sodium pumps, or to be more precise, without the Na,K-ATPase, is not possible. Animal cells need to maintain a high K+ and a low Na+ concentration in their cytoplasm as opposed to high Na+ and low K+ concentrations of their extracellular environment. The Na,K-ATPase creates electrochemical potential gradients for both ion species that are essential for cellular functions such as secondary active transport, excitability, and volume regulation. In 1957 the Na,K-ATPase was discovered by J. C. Skou (1), and since then there has been a wealth of studies published, providing increasing knowledge and insight into the structure and function of this active ion transporter of the plasma membrane at many different levels. Quite early the pump cycle was elucidated, the stoichiometry of 3 Na+/2 K+/1 ATP established, a consecutive or Ping-Pong-type mechanism (first Na+ out then K+ in) was demonstrated, and all these results were compiled into the famous Post-Albers cycle (2) that became the prototype of the reaction scheme for all P-type ATPases.

Molecular fibrils formed by aggregated amyloid peptides of various lengths and sequences are common pathological fingerprints of a large range of amyloid-related diseases, from type 2 diabetes to Parkinson’s, Huntington’s, or Alzheimer’s disease. Despite their tremendous biomedical importance, both the structural properties of peptide amyloid fibrils and the molecular mechanisms involved in their formation and propagation (i.e., the formation of nucleated seeds and fibril segments) remain notoriously difficult to investigate either experimentally or computationally (1–4).

Spines are tiny protrusions that densely stud the dendrites of neurons in the brain. Individual spines are the primary recipients of synaptic inputs from single axons, which emanate from other neurons in the central nervous system. A dendritic tree on one neuron may have hundreds of thousands of spines, making connections to a corresponding number of axons. The morphology of the spine typically consists of an ∼1-μm-diameter bulbous head connected to the dendritic shaft by a 50–150-nm-diameter × 500-nm-long cylindrical neck (Fig. 1). This morphology can be quite variable even on a single dendrite and has been characterized primarily by electron microscopy, because standard light microscopy approaches have insufficient resolution for these tiny structures. But, of course, electron microscopy precludes the analysis of spines in living tissue. To remedy this, a pair of articles in this issue of Biophysical Journal (1,2) combines two-photon and stimulated emission depletion (STED) microscopy to characterize individual spine morphologies on neuronal dendrites within freshly harvested live brain tissue (acute brain slices). They increase the resolution of two-photon excitation fluorescence microscopy from ∼350 nm to 60–80 nm, which is in the same range as the size of the spine necks (Fig. 1). Determining the size of the spine neck in living tissue will allow neuroscientists to explore how spines can behave as biochemical or electrical compartments to individually process synaptic inputs. Combining the virtues of two-photon microscopy with those of STED holds great promise for unraveling dynamic morphological changes of neuronal spines and their physiological role in the brain.

Biomolecules are finely tuned to perform their function in a precise range of temperatures. Variation of temperature outside this range, although not necessarily relevant to function, is a powerful way to explore structure and dynamics of these biomolecules. Well-known examples are the cooling of protein crystals to liquid nitrogen temperatures to investigate their x-ray diffraction patterns (1), and the physical trapping of short-lived reaction intermediates, e.g., by freeze-quenching methods (2) in mixed flows (3). Temperature changes can initiate or terminate biomolecular reactions by promoting or inhibiting energy barrier crossing, so that potential energy landscapes can be explored with adapted time-temperature protocols (4–7). The temperature cycle method consists in applying consecutive cycles with short heating and cooling steps (8,9), so that a dynamical process is decomposed into a series of snapshots, much as early movies decomposed the fast movements of galloping horses. By recording complete series of structural snapshots of a biochemical process, its whole dynamics can, in principle, be reconstructed. The temperature transitions should be as short as possible to limit unwanted transformations during heating and cooling periods. Because a structural variable can be measured at a low temperature over an arbitrarily long period, it can be determined more accurately than in short measurements at ambient temperature. Moreover, the method provides control of time resolution by tuning the duration and number of the high-temperature steps, and full control of barrier crossing rates by variation of the maximum and minimum temperatures in the cycle. However, the accurate and fast temperature changes required by this method cannot be achieved by conventional heating and cooling stages. Optical beams can induce highly localized and fast temperature gradients through heating of absorbing materials (8,10,11). Absorption of a laser pulse in a bulk material, for example water, leads to homogeneous heat release in the beam focus within picoseconds or nanoseconds. The cooling rate, however, is limited by heat diffusion out of the heated volume. This volume should therefore be reduced as much as possible to achieve short cooling times. Conventional ensemble spectroscopic methods cannot deal with very small sample volumes because of limitations in signal/noise. Single-molecule optics, however, are not liable to such volume limitations because they address only one molecule, independently of the surrounding sample volume. Single-molecule spectroscopy thus ideally complements temperature cycle techniques, as pointed out some years ago by Zondervan et al. (8). Those authors immerged their sample in a cold cryostat and focused an infrared laser onto a diffraction-limited spot of a metal film, thus demonstrating a laser-driven heating system with cycling times of some microseconds. Generalizing this method to aqueous environments is challenging because of phase transitions of water. Alternatively, raising the sample temperature brings other problems. For example, commonly used links for immobilization, such as the biotin-streptavidin couple, lose strength and can dissociate at elevated temperatures. Another problem is that many fluorophores may not withstand elevated temperatures, and properties such as fluorescence lifetime and quantum yield may be altered irreversibly. In this issue of the Biophysical Journal, Holmstrom et al. (12) describe a pulsed laser heating system that works in an aqueous environment by using water absorption for the fast heating process. Heat diffusion to the ambient environment remains responsible for the slower cooling step. The authors perform a detailed characterization of their temperature cycling system by monitoring the temperature-dependent fluorescence lifetime of Rhodamine B as a function of time and spatial position. They find that the temperature transitions between ambient (20°C) and maximum temperature (100°C) take milliseconds for picoLiter volumes. Because the heating temperature and duration can be precisely measured and controlled, they can minimize the dissociation of biotin-streptavidin by applying a proper temperature program. The authors also illustrate the power of temperature cycles by applying them to the reaction dynamics of DNA duplex dissociation (12). DNA duplex constructs include a reporter strand hybridized with a splint strand. Upon heating, the splint strand dissociates. When cooling again, the remaining reporter chain folds and hybridizes as a hairpin, bringing fluorescence donor and acceptor together and producing a FRET signal. DNA constructs that have reacted can be counted directly on the room-temperature image of the heated area. By monitoring the fraction of dissociated duplexes as a function of the duration and maximal temperature of the cycle, the authors propose a kinetic model for the dissociation reaction and deduce the energy barrier for duplex dissociation. The nonexponential dissociation kinetics is compatible with a multistep reaction requiring the unzipping of 5–6 basepairs before complete dissociation. From combinatorial arguments, the authors conclude that duplex fraying must take place simultaneously at both ends. Holmstrom et al. (12) demonstrate the combination of temperature cycles and single-molecule microscopy and apply it for, what we believe to be, the first time to provide detailed mechanistic information on a biomolecular reaction. To progress toward the dream of a complete mapping of potential energy landscapes for protein folding or for complex enzymatic reactions, shorter times and broader temperature ranges will be needed. This can be achieved with smaller sample volumes, stronger absorbers than water's vibrational overtone, and lower environment temperatures. This considerable experimental effort promises to be highly rewarding. Extending the application range of fast temperature cycles will open investigations of microsecond timescales, the stabilization of rare and short-lived intermediates, and eventually the elucidation of reaction pathways. Such a general and powerful method could tackle the dynamics and reactions of virtually all biomolecules, including enzymes (13), proteins (14,15), RNA (16), and their many complexes (17), at the single-molecule level.

Knowledge of membrane receptor organization is essential for understanding the initial steps in cell signaling and trafficking mechanisms, but quantitative analysis of receptor interactions at the single-cell level and in different cellular compartments has remained highly challenging. To achieve this, we apply a quantitative image analysis technique—spatial intensity distribution analysis (SpIDA)—that can measure fluorescent particle concentrations and oligomerization states within different subcellular compartments in live cells. An important technical challenge faced by fluorescence microscopy-based measurement of oligomerization is the fidelity of receptor labeling. In practice, imperfect labeling biases the distribution of oligomeric states measured within an aggregated system. We extend SpIDA to enable analysis of high-order oligomers from fluorescence microscopy images, by including a probability weighted correction algorithm for nonemitting labels. We demonstrated that this fraction of nonemitting probes could be estimated in single cells using SpIDA measurements on model systems with known oligomerization state. Previously, this artifact was measured using single-step photobleaching. This approach was validated using computer-simulated data and the imperfect labeling was quantified in cells with ion channels of known oligomer subunit count. It was then applied to quantify the oligomerization states in different cell compartments of the proteolipid protein (PLP) expressed in COS-7 cells. Expression of a mutant PLP linked to impaired trafficking resulted in the detection of PLP tetramers that persist in the endoplasmic reticulum, while no difference was measured at the membrane between the distributions of wild-type and mutated PLPs. Our results demonstrate that SpIDA allows measurement of protein oligomerization in different compartments of intact cells, even when fractional mislabeling occurs as well as photobleaching during the imaging process, and reveals insights into the mechanism underlying impaired trafficking of PLP.

The article by Marcovitz and Levy explores, using computer simulations, how obstacles facilitate DNA search by proteins (1xObstacles may facilitate and direct DNA search by proteins. Marcovitz, A. and Levy, Y. Biophys. J. 2013; 104: 2042–2050Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1). The observation that Lac repressor could find its preferred DNA binding site two orders-of-magnitude faster than expected from three-dimensional diffusion through solution was first made in the 1970s (2xThe Lac repressor-operator interaction. 3. Kinetic studies. Riggs, A.D., Bourgeois, S., and Cohn, M. J. Mol. Biol. 1970; 53: 401–417Crossref | PubMed | Scopus (451)See all References, 3xDiffusion controlled reaction rates in spheroidal geometry. Application to repressor-operator association and membrane bound enzymes. Richter, P.H. and Eigen, M. Biophys. Chem. 1974; 2: 255–263Crossref | PubMed | Scopus (193)See all References). Berg et al. (4xDiffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Berg, O.G., Winter, R.B., and von Hippel, P.H. Biochemistry. 1981; 20: 6929–6948Crossref | PubMedSee all References4) developed the theory that explained this rapid search in terms of a combination three-dimensional diffusion and sliding of the protein along the DNA chain. This theory is well accepted by the scientific community, but single molecule experiments have added a few caveats to the story, like the role of obstacles in the search (5xTowards a molecular view of transcriptional control. Zakrzewska, K. and Lavery, R. Curr. Opin. Struct. Biol. 2012; 22: 160–167Crossref | PubMed | Scopus (16)See all References, 6xVisualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Gorman, J., Plys, A.J...., and Greene, E.C. Nat. Struct. Mol. Biol. 2010; 17: 932–938Crossref | PubMed | Scopus (93)See all References, 7xSingle-molecule imaging reveals target-search mechanisms during DNA mismatch repair. Gorman, J., Wang, F...., and Greene, E. Proc. Natl. Acad. Sci. USA. 2012; 109: E3074–E3083Crossref | PubMed | Scopus (63)See all References, 8xAn end to 40 years of mistakes in DNA-protein association kinetics?. Halford, S.E. Biochem. Soc. Trans. 2009; 37: 343–348Crossref | PubMed | Scopus (115)See all References). For example, Gorman et al. (6xVisualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice. Gorman, J., Plys, A.J...., and Greene, E.C. Nat. Struct. Mol. Biol. 2010; 17: 932–938Crossref | PubMed | Scopus (93)See all References6) showed that protein diffusion along the one-dimensional DNA chain in chromosomes uses a hopping/stepping mechanism that readily bypasses stationary obstacles.Halford (8xAn end to 40 years of mistakes in DNA-protein association kinetics?. Halford, S.E. Biochem. Soc. Trans. 2009; 37: 343–348Crossref | PubMed | Scopus (115)See all References8) has argued that the enhanced search is due to electrostatics and that reduced dimensionality reduces the rate of target site location. Halford (8xAn end to 40 years of mistakes in DNA-protein association kinetics?. Halford, S.E. Biochem. Soc. Trans. 2009; 37: 343–348Crossref | PubMed | Scopus (115)See all References8) has also argued that one-dimensional diffusion is limited to regions of nearly 50 basepairs around each landing site. Taken together, these observations suggest that there are many factors combining to result in the fast location of a specific binding site. The inclusion of these factors into analytically solvable models is difficult and computational models are needed to gain insights. Here Marcovitz and Levy (1xObstacles may facilitate and direct DNA search by proteins. Marcovitz, A. and Levy, Y. Biophys. J. 2013; 104: 2042–2050Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1) use simple, structure-based, coarse-grained models and simulations to study the consequences that DNA coverage by obstacles has on the kinetics of one-dimensional searches on DNA. The simulations describe, in a simple way, the role of various effects, including a tuning between hopping and sliding due to the presence of salts, and the role of the mobility of obstacles on the search kinetics.Although all-atom simulations have been a preferred model to simulate biomolecular processes, this is one of many examples where the complexity of the problem does not allow explicit inclusion of all the detailed interrelated effects like hydration, diffusion, and specific binding. Marcovitz and Levy (1xObstacles may facilitate and direct DNA search by proteins. Marcovitz, A. and Levy, Y. Biophys. J. 2013; 104: 2042–2050Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1) and previous calculations by Vuzman et al. (9xFacilitated DNA search by multidomain transcription factors: cross talk via a flexible linker. Vuzman, D., Polonsky, M., and Levy, Y. Biophys. J. 2010; 99: 1202–1211Abstract | Full Text | Full Text PDF | PubMed | Scopus (38)See all References9) have shown that coarse-grained simulations capture the effects that many competing interactions have on the search for specific binding sites on DNA by a protein. Previous calculations by Vuzman and co-workers (9xFacilitated DNA search by multidomain transcription factors: cross talk via a flexible linker. Vuzman, D., Polonsky, M., and Levy, Y. Biophys. J. 2010; 99: 1202–1211Abstract | Full Text | Full Text PDF | PubMed | Scopus (38)See all References, 10xDNA search efficiency is modulated by charge composition and distribution in the intrinsically disordered tail. Vuzman, D. and Levy, Y. Proc. Natl. Acad. Sci. USA. 2010; 107: 21004–21009Crossref | PubMed | Scopus (62)See all References) have shown that intrinsically disordered tails in DNA binding proteins help the protein intersegmental transfer by a mechanism in which the protein jumps between distant DNA regions via an intermediate in which the disordered tail and the folded regions of the protein are transiently bound to different DNA segments. Givaty and Levy (11xProtein sliding along DNA: dynamics and structural characterization. Givaty, O. and Levy, Y. J. Mol. Biol. 2009; 385: 1087–1097Crossref | PubMed | Scopus (90)See all References11) also found that the efficiency of the search is significantly enhanced at high salt concentration due to a larger number of hopping events, with optimal searches for occurrences when the protein spends ∼20% sliding along the DNA and ∼80% hopping and performing three-dimensional diffusion, in absence of obstacles. In this issue, Marcovitz and Levy (1xObstacles may facilitate and direct DNA search by proteins. Marcovitz, A. and Levy, Y. Biophys. J. 2013; 104: 2042–2050Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References1) show that the effect of adding obstacles is not monotonic as a result from the competition between two main effects. That is, at low obstacle occupancy, the search is similar to the case of no obstacles; at very high concentration of obstacles, the protein has no accessibility to the DNA.At moderate occupancies (20%), there is a reduction in the effective one-dimensional diffusion coefficient (mean square deviation/time) relative to the coefficient for naked DNA. Interestingly, at slightly higher occupancies (34%) the one-dimensional diffusion coefficient is recovered. The authors conclude that the increase in obstacle occupancy may interfere with the optimal population of the search mechanism of 80% hopping, 20% sliding. They find that, for moderate occupancies, the one-dimensional search oversamples the linker regions and does not enhance the search, whereas for higher occupancies, the protein performs more hopping events.Although the specific values for the threshold occupancies (20 vs. 34%) and the percentage of hopping (80%) versus sliding (20%) depend on the specific, ad hoc, parameters and the simplicity of the model, the competition between these to effects may be real. Let’s wait and see what the experiments show.

Antimicrobial peptides are found in a large variety of organisms throughout all branches of cellular life: bacteria, archaea, and eukarya. They provide an evolutionarily ancient mechanism of defense against microbial invasion and play a fundamental role in the innate immunity of animals and humans. The initial discovery of Cecropin peptides in silk moth pupae, where they are expressed in response to insect injury and infection and during development (1), and the subsequent isolation of Magainin peptides from the skin of the African clawed frog, Xenopus laevis (2), established their importance as potent antibiotics (3). Early electrophysiological studies showed that Magainins, and other related peptides, cause highly selective and rapid lysis of bacteria in a concentration-dependent manner. The peptides are capable of forming pores that rapidly disrupt the electrical potential across membranes of bacterial cells, isolated mitochondria, and synthetic vesicles (3). Furthermore, the biological activities of antimicrobial peptides are highly synergistic; strong synergism is observed in their antimicrobial activity as well as in their cytotoxicity for other cell types, including transformed tumor cells (3). The finding that antimicrobial peptides synthesized from all D amino acids retain their full biological activity provided the first evidence that their function does not involve interaction of the peptide with a membrane receptor but instead relies on interaction of the peptide with the lipid bilayer membrane (4). This is consistent with the highly positively charged amphiphilic structures common to all peptides, which confer specificity for the predominantly electronegative membranes of bacteria. However, the functional mechanism of antimicrobial peptide activity has been a subject of intense discussion. In this issue, Strandberg et al. (5) describe the subtle effects of phospholipid structure on the interactions of antimicrobial peptides with membranes. Their results provide an explanation for the peptides' selective cytotoxicity for bacteria. Strandberg et al. (5) demonstrate that the interactions of both individual and synergized antimicrobial peptides with membranes depend not only on the electrostatic attraction between a cationic peptide and the anionic membrane surface, but also, very significantly, on the fundamental chemical structures of the phospholipids that influence their predisposition for membrane curvature. Negative curvature predisposition is observed in lipids with unsaturated hydrocarbon chains, while more ordered, saturated chains typically result in positive curvature (Fig. 1). Figure 1 Antimicrobial peptide (AMP) surface association is promoted by lipids with negative curvature predisposition. Lipids are shown with anionic headgroups (red) and hydrocarbon chains (yellow). The amphiphilic helical peptide, viewed down its helix axis, ... While, in some cases, antimicrobial peptides can insert across lipid bilayer membranes, their predominant conformation as membrane surface-associated α-helices was demonstrated early on by solid-state NMR structural studies (6,7). Membrane surface association has been elegantly rationalized as an essential early step in the functional mechanism of antimicrobial peptides, leading to massive pore formation and bacterial cell death. Rather than forming a classic channel with peptide helices inserted across the lipid bilayer membrane, the accumulation of peptides at the membrane surface is thought to cause a strain in the lipid bilayer, resulting in the formation of toroidal pores where the essential alignment of the peptide helices relative to the geometry of individual lipids is maintained (8–10). In a series of elegant solid-state NMR experiments Strandberg et al. (5) probe the membrane-associated conformations of two synergistic Magainin peptides, MAG2 and PGLa, as a function of phospholipid bilayer composition. The data unequivocally show that the membrane-surface-associated states of both isolated and combined MAG2 and PGLa are stabilized by phospholipids whose structures predispose them to negative membrane curvature. MAG2 and PGLa do not insert in lipid bilayers composed of negative curvature lipids with unsaturated hydrocarbon chains, regardless of the hydrocarbon chain length. By contrast, phospholipids with fully saturated dimyristoyl and dipalmitoyl hydrocarbon chains and a predisposition for positive curvature allow transmembrane insertion of PGLa and antagonize the surface-bound state. Notably, such lipids also fail to elicit appreciable membrane depolarization by the peptides (3), suggesting that the membrane inserted state may not be critical for function. The results obtained for membranes composed of bacterial phospholipids are particularly revealing. In the presence of bacterial lipids, with palmitoyl and oleoyl hydrocarbon chains and cardiolipin, phosphoethanolamine, and phospho-glycerol headgroups, both MAG2 and PGLa helices always align parallel to the membrane surface. These lipids have a marked propensity for negative membrane curvature and are enriched in bacterial membranes against which Magainin peptides exhibit their highest biological activity. Thus, the data suggest that membrane surface association of the peptides, promoted by negative curvature lipids, is a key aspect of their antimicrobial cytotoxicity. Recently, cardiolipin microdomains have been shown to localize to negatively curved regions of Escherichia coli membranes (11) and a clear phenomenological link has been demonstrated between anionic lipid clustering and the bacterial species specificity of several antimicrobial agents (12). The results of Strandberg et al. (5) help explain how lipid clustering could result in bacterial toxicity: the anionic and negative curvature properties of bacterial lipid clusters could conspire to recruit the accumulation of antimicrobial peptides and maintain them in a membrane surface orientation up to the critical concentration beyond which membrane disruption and massive pore formation are inevitable and commit the cell to death. Interestingly, the mitochondrial trans-acylating enzyme Tafazzin was recently shown to interact with specific pools of lipids that possess negative curvature properties (13). Just as proposed for Tafazzin, the data of Strandberg et al. (5) do not demonstrate that the peptides interact with curved membrane regions but, instead, how they interact with lipids that have a natural tendency to induce negative curvature in membranes. Antimicrobial peptides have been the focus of a large number of biophysical and biological studies aimed at understanding the molecular basis for their activity. Now, Strandberg et al. (5) provide significant new insights that can advance their development as effective therapeutics.

The analysis of the oligomerization of proteins is an old challenge that has never lost its importance. To the contrary, a growing number of proteins have been found to oligomerize under the influence of ligand binding as a key to biological control. Likewise, an increasing number of diseases are related to aberrant protein oligo- and polymerization. In a previous issue of the Biophysical Journal, an elegant method is presented to quantitatively characterize this phenomenon, based on the application of fluorescence correlation spectroscopy (FCS) (1). FCS measures fluctuations in concentrations of fluorescent molecules due to Brownian motion; as such, it needs small observation volumes and low concentrations of fluorescently labeled molecules (i.e., nanomolar as achieved in the observation volumes of contemporary confocal microscopes (2)). It is therefore useful to study protein associations at low concentration and allows for the determination of very high association constants. However, many proteins associate/dissociate in the micromolar or higher concentration regime. In FCS, this challenge can be overcome by using trace amounts of the fluorescently labeled protein in the presence of an excess of unlabeled protein, such that total protein concentrations much higher than nanomolar can be measured. An additional outcome of this scheme is that every oligomer contains only one label, so that the brightness remains the same for all oligomers, considerably simplifying analysis. This method has been applied to the oligomerization of tubulin (3), α-synuclein (4), tumor suppressor P53 (5), and many other proteins. Alternatively, brightness analysis—photon-counting histograms as well as other forms—has also been used extensively to study a number of protein systems (6–9). Typically, in these approaches all proteins are labeled and the brightness of each oligomeric species should be proportional to its size. These methods are powerful but do not always make use of the complementary diffusion information and still rely on nanomolar concentrations of fluorescent particles. In the article by Chakraborty et al., in a previous issue, a combination of the two techniques is presented by mixing labeled proteins with unlabeled ones at concentrations much higher than trace amounts. The method is used to elucidate the details of oligomerization of the AAA+ ATPase Rubisco activase (Rca), a protein whose self-association is important to its function. At low protein concentrations (between 50 and 300 nM), samples consisted of only labeled protein. FCS showed simple curves, with a diffusion coefficient (D1) independent of concentration and close to what was expected for monomer Rca. Photon-counting histograms also showed no changes in brightness, confirming that Rca is monomeric over this range of concentrations. However, at higher total protein concentrations (>550 nM), the FCS curves shift to longer timescales with increasing protein concentration, indicative of the oligomerization of the protein. Despite the presence of multiple oligomeric species at these concentrations, the FCS curves could be fit adequately with an equation for a single diffusing species, yielding an apparent diffusion coefficient (Dapp), which represents a brightness-weighted-average diffusion coefficient of all species present. Dapp was found to decrease in a continuous manner with increasing concentration, as has been observed previously for tubulin (3), α-synuclein (4), and p53 (5). Because of this, the relative concentrations of each of the oligomeric species could not be determined from simply fitting the autocorrelation curves. Thus, the authors developed a method of analysis using Dapp/D1 to determine the average size of the oligomers at each concentration of protein. The relationship between oligomeric size and diffusion coefficient was modeled in two ways: 1. Assuming spherical symmetry, which leads to Dk/D1 = k−1/3 where k is the number of monomers in the oligomer, or 2. based on the calculation of the radius of gyration using x-ray crystallographic data. It is worth noting that the first case is only approximately correct because a spherical dimer cannot have a radius that is 2× that of a monomer, and that for irregular shapes, D can also be calculated using the Hydrosub program (10). (The effect of shape on D is extensively discussed in the Supporting Material of the article by Chakraborty et al. (1).) For further analysis, the authors derived an autocorrelation function to describe their polydisperse sample. This required assuming a well-defined diffusion coefficient and brightness for each oligomeric species, thus taking into account the fraction of labeled protein and the random distribution of labeled protein among the various oligomers. By estimating the Dk for each oligomer from x-ray crystallography data, the authors develop a fitting function where the only unknown parameter is the concentration of each oligomer, Ck, dependent upon the total protein concentration and the dissociation constants describing the equilibrium behavior of the oligomers. Dapp/D1 was plotted as a function of total protein concentration and this data was fit to their model. While a number of different scenarios are discussed, the best fit that could be obtained assumed the formation of dimers, tetramers, and hexamers with dissociation constants with 4, 1, and 1 μM, respectively, with the association into even larger species at very high protein concentrations (>10 μM). Although hexamers have been identified previously by electron microscopy and mass spectrometry, and SE-HPLC elution profiles indicate a mix of oligomeric species, the measurements described here allowed for an analysis of the stoichiometry of the various oligomers that was previously unobtainable. This method should allow the quantitative study of allosteric regulation of Rca, which is of importance in the context of biological carbon fixation. This method is very general and the equations are transparent and should have wide application to in vitro measurements. Its usefulness for measurements in the cell is, however, limited because concentrations cannot be varied at will, and the relation between size and diffusion coefficient is more complex in this crowded environment. Moreover, there are the known difficulties with the definition of the confocal volume in the inhomogeneous cellular environment (11). However, image correlation spectroscopy using brightness information seems to be able to give information on oligomer distributions (12,13) and heterospecies partition analysis for mixtures of different proteins (14).

Reference EPFL-ARTICLE-208509doi:10.1016/j.bpj.2014.12.002View record in Web of Science Record created on 2015-05-29, modified on 2017-05-12

The mechanism that makes a eukaryotic flagellum or cilium beat is one of cell biology's most enduring puzzles. As long ago as 1835, Dr. W. Sharpey (1xCilia. Sharpey, W. : 606–638See all References1) noted that the beating of cilia is a confirmed biological phenomenon that warrants further study. Today, we know that the motor protein dynein powers the bending of cilia and flagella. Dynein does this by creating traction between the outer doublets of the microtubule scaffolding called the axoneme that is at the heart of all cilia and flagella. The article by Dr. Charles Brokaw (2xSimulation of cyclic dynein-driven sliding, splitting and reassociation in an outer doublet pair. Brokaw, C.J. Biophys. J. 2009; 97: 2939–2947Abstract | Full Text | Full Text PDF | PubMed | Scopus (16)See all References2) in this issue of Biophysical Journal provides new insight into the mechanism of the interaction that generates the flagellar and ciliary beat. The study provides pivotal information on the nature of the dynein tubulin interaction, and is useful to discriminate between the different proposals regarding the mechanism of ciliary and flagellar beating.One of the more elegant experiments investigating the interaction of dynein and the doublet microtubules in the flagellum was conducted by Aoyama and Kamiya (3xCyclical interactions between two outer doublet microtubules in split flagellar axonemes. Aoyama, S. and Kamiya, R. Biophys. J. 2005; 89: 3261–3268Abstract | Full Text | Full Text PDF | PubMed | Scopus (41)See all References3). They showed that individual doublets of “frayed” flagellar axonemes of the green algae Chlamydomonas reinhardtii occasionally will reassociate with each other and establish a kind of beat-like cycle. The doublets adhere to each other, bend, and subsequently separate from each other, from the base to the tip, and then repeat the sequence. This outer-doublet microtubule pair is the simplest flagellar fragment that still shows the ability to construct beat-like oscillations. Therefore, it is likely that it is telling us something important about the oscillation mechanism underlying the beating of the whole flagellar structure. Dr. Brokaw uses images from published studies of the Kamiya group and applies the best biophysical modeling tools to simulate these classic experiments and elucidate the physics of the two-doublet system.Currently, there are three rather different views of how flagella and cilia convert the action of the dynein motors into a rhythmic beat. The first view is that a complex of regulatory proteins residing on the central pair and radial spokes actively sends signals to the dynein motors through a dynein regulatory complex located on the surface of the outer doublets. It is hypothesized that a regulatory enzymatic cascade originates at the central pair-spoke interaction sites and acts to turn the dyneins on and off in the proper order to produce beating (4xRegulation of flagellar dynein by the axonemal central apparatus. Smith, E.F. Cell Motil. Cytoskeleton. 2002; 52: 33–42Crossref | PubMed | Scopus (65)See all References4). The second view maintains that the dynein molecules themselves are tuned oscillators that can be entrained to synchronize their cross-bridge cycles into waves of activity under the appropriate loading conditions present in the intact flagellum (5xHow molecular motors shape the flagellar beat. Riedel-Kruse, I.H., Hilfinger, A., and Julicher, F. HFSP J. 2007; 1: 192–208Crossref | PubMedSee all References5). The third view contends that the dyneins are engaged and disengaged by changes in the interdoublet spacing as the axonemal structure is distorted by stress (6xA model for flagellar motility. Lindemann, C.B. and Kanous, K.S. Int. Rev. Cytol. 1997; 173: 1–72Crossref | PubMedSee all References6). This last view is called the “geometric clutch” hypothesis.The simulations conducted by Dr. Brokaw indicate that the oscillations of two doublets isolated from the intact flagellum can be explained satisfactorily by a mechanism whereby the dyneins detach from their binding sites when the transverse stress between the doublets exceeds the capacity of the dyneins to hold on, as illustrated in Fig. 1Fig. 1. This supports the contention that dynein action can be regulated by the transverse force (t-force) that develops between adjacent doublets. If this mechanism of oscillation also functions in the intact axoneme, it would lend support to the geometric clutch interpretation of the flagellar beat. This does not rule out the possibility that the other major hypotheses could also help explain the generation of the beat, but it does lend credibility to the hypothesis that t-force between the doublets can modulate the activity of dynein. It also tells us that the flagellum possesses a means of generating repetitive cycles of oscillation that is independent of the influence of the central apparatus. The switching mechanism in the two-doublet experiments appears to be more dependent on the t-force acting on the dyneins than on the cargo load the dyneins are carrying.Figure 1Proposed mechanism of dynein dissociation in the two-doublet experiment. (A) Two adjacent doublets associate by dynein attachment. Dynein motor activity results in compression of the upper doublet and tension on the lower doublet. (B) The doublet under compression undergoes buckling, which creates a curvature and t-force. (C) The t-force increases and overcomes the adhesion of the dyneins to produce a spreading wave of detachment.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe authors of the original experimental report (3xCyclical interactions between two outer doublet microtubules in split flagellar axonemes. Aoyama, S. and Kamiya, R. Biophys. J. 2005; 89: 3261–3268Abstract | Full Text | Full Text PDF | PubMed | Scopus (41)See all References3) dismissed the geometric clutch interpretation of their results as inconsistent with their observations. This was based on the fact that the doublets are often seen to separate when there is no initial curvature, and curvature is necessary for the development of transverse stress. Dr. Brokaw specifically addresses this concern in his analysis and shows that the buckling instability of the member of the doublet pair that is under compression creates a local curvature, as illustrated in Fig. 1Fig. 1, and that the resultant t-force acting on the dynein increases rapidly due to this dynamic instability.The computational approach used by Dr. Brokaw provides new and valuable quantization of the dynein-tubulin interaction and has the potential to be of value in developing an accurate model of flagellar and ciliary functioning. By fitting the modeled simulations to the experimental data, the analysis gives us a first estimate of the adhesion force with which real dynein motors hold on to the microtubule to which they are bound. It also provides a first look at the kinetics of disassociation and reassociation of the motors with their binding sites. These quantitative parameters of adhesion, disassociation, and reassociation of dynein with tubulin are elements of the geometric clutch model, but to date they have not been amenable to experimental study or to quantization. Dr. Brokaw's approach allows useful information to be mined from the two-doublet studies. Ultimately, this will permit a much better understanding of the kinetics of dynein tubulin interaction and the mechanism of flagellar beating.

Akin to thepuckon an ice hockey rink,the lateral dynamics of a representativemembrane protein exhibits rich be-havior(1).Dependingontheinteractionwiththehockeyplayersandotherobsta-cles (membrane constituents), the puck(protein)candart aroundthe ice(mem-brane surface) or be stalled momen-tarily (transient confinement zone). Ifwe watched the motion of a singlepuck, we would need to watch manyhockey games beforewe might eventu-allyworkouttherulesofthegame.Butwhat if we could watch many games inparallel? This is where the suite of im-age correlation spectroscopy (ICS)techniques and its newest member, k-space image correlation spectroscopy(kICS) comes in Abu-Arish et al. (2).ICSandkICSaremembersofafam-ily of techniques which began withfluorescence correlation spectroscopy(3). Fluorescence fluctuation analysisprovides the statistical mechanicalfoundation with correlations betweenfluorescence fluctuations measuredrelative to a given lag vector. Correla-tions can be made as a function oftime (fixed space) called FCS (3), fluc-tuationsinspace(fixedtime)calledICS(4), and space-time fluctuations calledSTICS (5). The ergodic principle en-sures equivalence between occupancyfluctuations whether in time or space.In temporal ICS or TICS, one essen-tially images in time (using a laserscanning fluorescence confocal micro-scope) many molecules in parallel asthey change positions from one imageto the next image. If the molecules donot move between the first image andthe second image, the spatial correla-tion between image one and imagetwo will be high. However, if move-ment does occur between image oneand imagetwo,there isaloss of spatialcorrelation. By analyzing the correla-tion between images collected atdifferent times, information on proteinmotioncouldbeobtained.Thismethodprovidesinformation from manymole-cules without the need for tracking in-dividual particles. A problem endemicto all these methods is that fluctuationsin fluorescence are measured asopposed to particle fluctuations whichare the desired quantities in evaluatingtransport properties.In kICS (6), images collected from atime-seriesarefirstFouriertransformedinto k-space before image cross-corre-lation is performed. This has twodistinct advantages over (real space)ICS. First, fluctuations from photophy-sics (e.g., cis-trans isomerization, (de)protonation, reversible dark statequenching, reversible photobleaching)do not contribute to the determinationof transport coefficients (6). Second,determinationofthelateralpointspreadfunction dimension is not required, un-like conventional fluorescence fluctua-tion approaches (6).In this issueof the Biophysical Jour-nal, the pioneer of ICS and kICS, PaulWisemanhasteamedupwithAsmahanAbu-Arish,ElvisPandzic,JulieGoepp,Elizabeth Matthes, and John Hanrahan(2) to gain new insights into the dy-namics of a biomedically importantmembrane protein, the cystic fibrosistransmembrane conductance regulator.Using the kICS technique, the authorshaveprovidedevidencefortwopopula-tions of cystic fibrosis transmembraneconductance regulator (CFTR) mole-cules, which differed in degree ofconfinement and lateral motion on thecell surface. Impressively, they wereable to extract information on the dy-namics of CFTR inside domains,CFTR dynamicsoutside(andbetween)domains, fractional populations, anddegree of confinement (within do-mains). ICS analysis delivered clusterdensities and mean number of mole-cules per cluster.The authors examined the effect ofcholesterol on dynamics and clusteringbehavior of CFTR. Cholesterol has aremarkable effect on membrane pro-tein assembly, dynamics and function(7–9). Depletion of cholesterol causedthe confinedfractionand averagenum-ber of CFTR molecules per cluster todecrease, whereas increase in choles-terol were found to be associated withincrease in clustering and increasedconfined fraction. Interestingly, viralinfection was shown to increase clus-tering further into larger platformswith reduced CFTR mobility. Theseobservations and analyses suggestthat cholesterol-influenced membranedomains play an important role in thecell surface behavior and pathologyof CFTR.Aside from the important new in-sights into this anion channel, the re-sults of this study revealed howcomplex cell surface dynamics andclustering can be measured usingpowerful fluorescence microscopytechniques. With these new methodsin hand, biophysicists can sit back,relax and enjoy the hockey game.REFERENCES

The development of an organism is a fascinating showdown of biology’s might, where just a single fertilized cell gives rise to all cells that together build the multifaceted parts of an organism. An elaborate biological machinery of proteins and metabolites works together to specify individual cell positions and cell identities. How could the simple rules of physics assist the dynamics of development? The physical forces that minimize surface tension have proven to be very successful in determining cell position within tissues (1). Cell shape and arrangement (2) and cell sorting (3) result from the balance of surface forces generated from cell properties such as cell contractility and cell-cell adhesion. In this issue, Struntz et al. (4) show that the forces arising from elastic repulsion between cells account for the cell positions during the first cycles of embryogenesis of the nematode (roundworm), Caenorhabditis elegans. The development of nematodes is characterized by cells acquiring their unique identity at an early stage. In fact, the entire cell lineage is invariant between different eggs and fully documented for C. elegans (5). Cells acquire their different identities by the unequal distribution of some factors during asymmetric cell division. In early embryogenesis, cell identity is also specified by a cell’s interaction with its immediate neighboring cells. Therefore, the positions of cells within the egg shell are important in defining cell identity and ensuring successful development. How does the embryo achieve a robust and reliable positioning of cells? Struntz et al. (4) use gentle yet fast, three-dimensional fluorescence microscopy by employing selective plane illumination microscopy to trace the trajectories of individual cell nuclei during early embryogenesis. Variation of the nuclei positions between individual eggs is observed to be 12), the repulsive forces alone are found to be insufficient to reliably determine cell nuclei position. At this stage, the packing problem becomes mathematically difficult and may have multiple equally good solutions, so guidance by biological signaling is needed for direction. The work by Struntz et al. (4) provides a fascinating physical framework that can, in future, be complemented with the observation and perturbation of biological factors to study how biochemical signaling and mechanical forces are intertwined to coordinate development. During the last decade, surface forces have been found to assist in multiple processes during the development of organisms (7). Struntz et al. (4) now show that the elastic forces of mutual repulsion between cells are enough to explain the robust positioning of cells during the early stages of C. elegans embryogenesis. This is an elegant example of simplicity in the midst of the bewildering complexity of development.

In this issue, Willenbrock et al. (1)address how leukocytes achieve decel-eration during rolling and arrest onendothelium in the vasculature neartissue sites of inflammatory insult.While cell capture from the bloodstream and rolling is largely the func-tional domain of selectin adhesionreceptors that are constitutively ex-pressed on leukocytes and rapidlydeployed on endothelium to bindglycosylatedligands,theyarenotsuffi-cient to decelerate leukocytes. This isthe function of integrins, which aretransmembrane heterodimers consist-ing of a large head on two legsthat extend during cell activation tomediate ligand binding (2). Most ofthe heterodimer is expressed extracel-lularly, but both subunits traverse theplasma membrane and terminate inshort cytoplasmic domains wherethey dynamically assemble with cyto-skeletal and signaling complexes.Thus, ligand binding via integrinsfacilitates cell arrest in the stiff shearforce of blood flow and serves tobridge the extracellular milieu to theintracellular cytoskeleton—a criticalstep for initiating transendothelialmigration (3). Two decades haveelapsed since natural mutations werediscovered in the genes coding forintegrin and selectin receptors thatcause human leukocyte adhesion defi-ciency disease, LAD-I and LAD-II,respectively (4). Intense study of therelationship between structure andfunction has revealed that integrinseffectively serve as gatekeepers thatcontrol access of leukocytes by under-going an allosteric shape shift andlaterally redistribute on the membraneto modulate the affinity and valenceinbindingtoIg-superfamilycounterre-ceptors including ICAM-1 upregulatedon inflamed endothelium. Bondforma-tion on the outside has direct influenceon integrin linkage on the inside to thecortical cytoskeleton that in part deter-mines bond lifetime and adhesivestrength.The most recent immunodeficiencyassociated with impaired leukocyterecruitment is the LAD-III phenotype,characterized as a loss of functionmutation in Kindlin3, the predominantisoform expressed in leukocytes (5).This adaptor molecule is necessaryfor triggering the allosteric shift in in-tegrins and a rapid increase in densityof focal clusters on the plasmamembrane necessary for binding toICAM-1 in sufficient numbers todecelerate the cell. A recent flurry ofstudies have focused on the role ofKindlin3inintegrinfunction,revealingthat its participation in postcaptureadhesive events involves associationand the b-subunit cytoplasmic domainof LFA-1 and the cortical cytoskeletonvia talin (6). Evidence of the associa-tion of Kindlin3 with the cortical cyto-skeleton only provides a static readoutof how the brakes are deployed duringleukocyte touchdown and taxiing tosites of transmigration. To evaluatethe complex and overlapping mole-cular binding events and discriminatetheir respective contributions to decel-eration and arrest, it is necessary tocarefully track the position andvelocity of cells as a function of time.An automated solution, denoted‘‘Leukotrack’’, was developed byWillenbrock et al. to identify leuko-cytes captured by video microscopywhile rejecting streaks and smalldebristhatotherwisecanbiastheaccu-rate analysis of cell trajectory undershear flow. This program facilitatesanalysis of the diverse kinematics ex-hibited by leukocytes during exposureto hydrodynamic shear while interact-ing on a substrate. By storing theposition of each cell using a nearest-neighbor image analysis algorithm,Leukotrack effectively discriminatesrolling from arrested and migratingcell behaviors and facilitates a statis-tical comparison that otherwise wouldbe laborious, mind-numbing work.To address the specific defects inleukocyte rolling and deceleration toarrestinLAD-IIIpatients,Willenbrocket al. (1) employed Epstein Barr virus-transformed B lymphoblastoid cellsderived from peripheral blood mono-nuclear cells of Turkish LAD-IIIpatients. The adhesive behavior oftransformed B cells was comparedbetween two patients and parentEpstein Barr virus-transformed cells.B-cell rolling on E-selectin induced adistinct activation signal to shiftLFA-1toahighaffinitystatethatbindstightlytoitscognateendothelial ligandICAM-1 under shear flow. This tech-nique made possible direct imagingof LFA-1-mediated adhesion of B cellsand the Leukotrack program providedacquisition of kinetic data fromhundreds of cell trajectories, therebyfacilitating discrimination of the roleof Kindlin3 in LFA-1-mediated activa-tion and its participation in the multi-step process of recruitment.A key question addressed by theanalysis was how dynamic signalingvia E-selectin ligation allostericallytriggers the extension of LFA-1 andupshift from low to an intermediateand high affinity conformation througha mechanosensory mechanism pro-moted by shear-induced bond tension.Recent data from my laboratory andothers indicate that coupling ofLFA-1 to the actin cytoskeleton isrequired for high affinity and mul-tivalent binding to heterodimericICAM-1 (3,7). Remaining unresolvedis how Kindlin3 functions in leukocytedeceleration in the context of associa-tion with the cytoplasmic domain of

In this issue of the Biophysical Journal, Drocco et al. describe a new approach to studying the biophysics of membrane gradient polarization that is certain to provide new insights into the cellular polarization that define morphogenetic axes during embryogenesis. In a seminal 1970 Nature article (1xDiffusion in embryogenesis. Crick, F. Nature. 1970; 225: 420–422Crossref | PubMed | Scopus (405)See all References1), and even before a clear quantitative basis of molecular diffusion was achieved, Francis Crick conjectured that “a simple order-of-magnitude calculation suggests that diffusion may be the underlying mechanism in establishing morphogenetic gradients in embryonic development”. Crick based his work on the concept previously proposed by C. M. Child (2xPatterns and Problems of Development. Child, C.M. CrossrefSee all References2) and L. Wolpert (3xPositional information and the spatial pattern of cellular differentiation. Wolpert, L. J. Theor. Biol. 1969; 25: 1–47Crossref | PubMedSee all References3) that molecular morphogen gradients define axes of embryonic development. Crick recognized that if you had a site of localized morphogen insertion, that the slow diffusion coefficients of these molecules would, because of the large size of these cells, maintain a molecular gradient for extended periods of time, times sufficient to play a role in embryogenesis.Crick's principal interest was in multicellular gradients. However, in the late 1970s and early 1980s, it became clear that single cells in early embryogenesis establish molecular gradients along axes that define important cleavage planes (4xChanges in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo. Ducibella, T., Ukena, T...., and Anderson, E. J. Cell Biol. 1977; 74: 153–167Crossref | PubMedSee all References, 5xDistribution of antibody- and lectin-binding sites on dissociated blastomeres from mouse morulae: evidence for polarization at compaction. Handyside, A.H. J. Embryol. Exp. Morphol. 1980; 60: 99–116PubMedSee all References, 6xCytoplasmic polarity develops at compaction in rat and mouse embryos. Reeve, W.J.D. J. Embryol. Exp. Morphol. 1981; 62: 351–367PubMedSee all References, 7xMembrane events associated with the generation of a blastocyst. Johnson, M.H. Int. Rev. Cytol. Suppl. 1981; 12: 1–37PubMedSee all References). In the mouse, a radial axis is established during compaction at the 8-cell stage and manifests itself in the establishment of gradients in both membrane and cytoplasmic components. Ziomek and Johnson (8xCell surface interaction induces polarization of mouse 8-cell blastomeres at compaction. Ziomek, C.A. and Johnson, M.H. Cell. 1980; 21: 935–942Abstract | Full Text PDF | PubMedSee all References8) demonstrated that cell-cell contact between 8-cell murine blastomeres led to polarization of concanavalin A surface receptors, with subsequent cleavage 8-cell–16-cell occurring perpendicular to the axis defined by the gradient. This cleavage defines the earliest cellular differentiation into two cell types within the subsequent blastocyst: the inner cell mass, which becomes the embryo, and the trophectoderm, which becomes the amniotic sack and placenta.Handyside et al. (9xPolarized distribution of membrane components in two-cell mouse embryos. Handyside, A.H., Edidin, M., and Wolf, D.E. Rouxs Arch. Dev. Biol. 1987; 196: 273–278Crossref | Scopus (3)See all References9) observed a similar polarization of antigens, lectin receptors, and lipids opposite to the previous cleavage furrow at the 2-cell stage. Polarization appeared during the first 4–5 h after first cleavage and diminished, but did not disappear over the remaining period before second cleavage. This diminution was consistent with what would be predicted based upon fluorescence recovery after photobleaching measurements of membrane component lateral diffusion (10xChanges in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: indications from the lateral diffusion rates of fluorescent lipid analogs. Wolf, D.E., Edidin, M.E., and Handyside, A.H. Dev. Biol. 1981; 85: 195–198Crossref | PubMed | Scopus (19)See all References10). Thus localized morphogen insertion, coupled with known diffusion rates, were sufficient to support morphogenetic gradients until next cleavage.In their study, Handyside et al. (9xPolarized distribution of membrane components in two-cell mouse embryos. Handyside, A.H., Edidin, M., and Wolf, D.E. Rouxs Arch. Dev. Biol. 1987; 196: 273–278Crossref | Scopus (3)See all References9) assumed that newly inserted membrane material persisted over the entire cleavage-to-cleavage interval. However, more generally, if one assumes localized insertion of new membrane, the persistence of morphogenetic gradients, as well as the degree of polarity and gradient steepness, will depend upon the interplay of several factors: 1), diffusion coefficient, 2), the presence or absence of a corresponding sink at the other pole of the cell, 3), the lifetime of the morphogen at the cell surface, and 4), the rate of synthesis and insertion of new morphogen.In this issue, on pages 1807–1815, Drocco et al. introduce a new and powerful tool for assessing the effects of protein lifetime on morphogenic gradients. Coupled with imaging techniques, such as confocal microscopy, their approach enables a much more complete mathematical treatment, and therefore the ability to distinguish between models of the gradient problem, which differ in the relative effects of the four different factors discussed above. Drocco et al. have generated a fusion between the Drosophila embryo morphogen Bicoid (Bcd) (11xA gradient of bicoid protein in Drosophila embryos. Driever, W. and Nusslein-Volhard, C. Cell. 1988; 54: 83–93Abstract | Full Text PDF | PubMed | Scopus (555)See all References, 12xThe bicoid protein determines position in vthe Drosophila embryo in a concentration-dependent manner. Driever, W. and Nusslein-Volhard, C. Cell. 1988; 54: 94–105See all References) and the photoswitchable protein Dronpa (13xReversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Habuchi, S.R., Ando, R...., and Hofkens, J. Proc. Natl. Acad. Sci. USA. 2005; 102: 9511–9516Crossref | PubMed | Scopus (276)See all References13). Dronpa can be switched from its bright state into its dark state using a 496 nm Argon laser and then reconverted back to the bright state using a 405 nm diode laser. Photoconversion between the dark and light states of Dronpa, effectively allows tagging the protein present at a given point in the cell cycle and then following its subsequent evolution as well as the presence or absence of newly inserted material. They have found that Bcd lifetime changes from 50 min before the 14th mitotic cycle to only 15 min during cellularization. In addition, photoswitching of the Dronpa-Bcd fusion enables systematic mimicking of increased Bcd degradation, enabling experimental determination of the effect of morphogen lifetime on gradient shape and persistence.Because it is based on standard and widely available fusion and photoswitching techniques, the method of Drocco et al. is broadly applicable to the study of morphogens and morphogenetic gradients in a multitude of embryonic models. The method coupled with fluorescence recovery after photobleaching or fluorescence correlation spectroscopy measurements of morphogen diffusion coefficients enable a clear and definitive answer, in both biophysical and biochemical terms, to the problem of morphogenetic gradients first posed by Frances Crick (1xDiffusion in embryogenesis. Crick, F. Nature. 1970; 225: 420–422Crossref | PubMed | Scopus (405)See all References1) more than 40 years ago.

We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency and location along filaments. The emergent behavior of mechanically heterogeneous filaments, particularly under confinement, emphasizes that severing in cells is likely to be influenced by multiple physical and chemical factors.

The central principle justifying continued investment in structural genomic projects has been founded upon the assumption that the resolution of static three-dimensional structures of a finite number of proteins will provide the key to understanding biological activity. Over the last decade, biophysical characterization, allied with computer-based prediction, has convincingly shown that >30% of proteins present in the human proteome may contain continuous regions of at least 30 amino acids in length that are intrinsically disordered. This realization highlights a detail that may in hindsight appear evident; that the determination of the three-dimensional structure of folded globular domains, while essential, represents only one aspect of the quest to complete the proteomic structural map. The role that disorder plays in such proteins and the advantages it confers in terms of function are subjects of intense interest and debate, whose resolution represents one of the major challenges for contemporary structural biology. (1xIntrinsic disorder and functional proteomics. Radivojac, P., Iakoucheva, L.M...., and Dunker, A.K. Biophys. J. 2007; 92: 1439–1456Abstract | Full Text | Full Text PDF | PubMed | Scopus (405)See all References1) In some cases, disordered regions provide entropic linkers or spacers, whose role appears to be to position different, possibly folded domains in three-dimensional space. Such multidomain systems with folded domains separated by flexible linkers are particularly prevalent in transcription and replication, cellular signaling and adhesion, and in viral proteins. The behavior of linker regions and the relationship between primary sequence and different levels of flexibility encoded in their primary sequence remain open and poorly understood aspects of structural biology. (2xNMR studies of modular protein structures and their interactions. Pickford, A.R. and Campbell, I.D. Chem. Rev. 2004; 104: 3557–3566Crossref | PubMed | Scopus (44)See all References2) There is therefore a compelling need for novel methodology to develop a full molecular understanding of these complex proteins.Due to their very high degree of conformational flexibility, this is of course far from trivial. The most common approaches evoke explicit ensembles of rapidly interconverting conformers, based on agreement with extensive sets of experimental data measured in solution. NMR spectroscopy is without doubt the tool of choice for studying both local and long-range structural propensities at atomic resolution in intrinsically disordered proteins, while small angle scattering (SAS) provides essential information about overall molecular dimensions (3xStructural characterization of flexible proteins using small-angle x-ray scattering. Bernado, P., Mylonas, E...., and Svergun, D.I. J. Am. Chem. Soc. 2007; 129: 5656–5664Crossref | PubMed | Scopus (495)See all References3). The combination of complementary experimental NMR and SAS data with ensemble molecular descriptions has indeed allowed the characterization of entire proteins involving significant folded and unfolded domains (4xQuantitative determination of the conformational properties of partially folded and intrinsically disordered proteins using NMR dipolar couplings. Jensen, M.R., Markwick, P.R.L...., and Blackledge, M. Structure. 2009; 17: 1169–1185Abstract | Full Text | Full Text PDF | PubMed | Scopus (112)See all References4). NMR data report on population-weighted averages of all conformations exchanging on timescales faster than the millisecond range, but ensemble descriptions of disordered chains rarely address the important question of dynamic interconversion rates beyond this simple limit. The dynamics and thermodynamics of conformational change are, however, of great importance if we are to develop a true statistical mechanical representation of the protein in solution.NMR spin relaxation can, in principle, provide an answer to this question, by characterizing the mobility of interatomic bonds throughout the protein on timescales in the hundreds of picosecond to tens of nanosecond range. The interpretation of relaxation rates in disordered chains, or in domains connected by flexible linkers, is complicated by the potential coupling between motion of individual domains and overall motion of the entire multidomain protein. It has been shown, however, that if a molecular description of the protein ensemble is available, combination with relaxation data can provide information about reorientational eigenmodes present in the ensemble. (5xGeneral framework for studying the dynamics of folded and nonfolded proteins by NMR relaxation spectroscopy and MD simulation. Prompers, J.J. and Bruschweiler, R. J. Am. Chem. Soc. 2002; 124: 4522–4534Crossref | PubMed | Scopus (121)See all References5) Timescales relevant to conformational interconversion can thereby be derived directly from the relaxation data.In this groundbreaking study, the authors combine many of these approaches to study ribosomal protein L12, a two-domain protein that dimerizes via its N-terminal domain (NTD). (6xStructure and dynamics of ribosomal protein L12: an ensemble model based on SAXS and NMR relaxation. Bernado, P., Modig, K...., and Akke, M. Biophys. J. 2010; 98: 2374–2382Abstract | Full Text | Full Text PDF | PubMed | Scopus (71)See all References6) NTD is separated from the C-terminal domain (CTD) via a flexible 20-residue chain such that the protein can be described in terms of three folded domains, connected by a more flexible linker. The flexibility of the CTD has been related to control of ribosomal function. The authors use molecular modeling to sample the conformational space available to the three domains as extensively as possible. This sampling is expressed in terms of 10,000 dimeric conformers (derived from 2 million original conformers) selected on the basis of agreement with experimental SAS data. This ensemble contains the conformational distributions of interdomain distances, and defines the volume space occupied by the protein. Although it contains no information about the dynamic timescales, it is assumed, reasonably, to contain the reorientational properties of the protein. Timescale information can be provided from the NMR relaxation data analysis of the individual domains within the ensemble. The combined analysis reveals anticorrelated motions of the CTD and NTD dimer and provides novel insight into the behavior of the linker regions in solution. The authors also mention an important issue concerning the resolution of this kind of ill-defined inverse problem, where the number of conformational degrees of freedom far outweighs the ability of the experimental data to uniquely restrain the system: The approach relies on the ability of the initial analysis to define a relevant ensemble of structures that accurately represents the behavior of the protein in solution. The next challenge lies in the development of robust validation techniques (some kind of R-factor) that will allow the estimation of levels of confidence in the proposed models.The study clearly demonstrates how the complementary dependences of SAS and NMR relaxation data can be combined to extract information that is far beyond the sum of their parts. Similar approaches can be used to combine different types of NMR data, for example dipolar couplings, chemical shifts, or paramagnetic enhancements (4xQuantitative determination of the conformational properties of partially folded and intrinsically disordered proteins using NMR dipolar couplings. Jensen, M.R., Markwick, P.R.L...., and Blackledge, M. Structure. 2009; 17: 1169–1185Abstract | Full Text | Full Text PDF | PubMed | Scopus (112)See all References4). Developments such as these are providing the blueprints for future studies of multidomain, highly flexible, and intrinsically disordered proteins, and thereby establishing the basic tools for completing our understanding of the conformational proteome.

How do isolated cells reach out collectively to remodel the environment in which they reside? We have known for some time that in certain processes, such as wound-healing, cells appear to act together, contacting one another through bands of remodeled collagen stretching over distances that are very long compared to the size of a cell (Fig. 1, left panel). We also know that fibroblasts act locally, synthesizing and remodeling extracellular matrix (ECM) proteins in response to signals that are only partially understood (Fig. 1, right panel).

Human life depends on the fact that blood remains fluid while it is in the vessels, but clots promptly when released by injury. Nature accomplishes this balancing act by means of a collection of a dozen or so proteases and cofactors, arranged in a reaction network, as described formally in Panteleev et al. (1xTask-oriented modular decomposition of biological networks: trigger mechanism in blood coagulation. Panteleev, M.A., Balandina, A.N...., and Ataullakhanov, F.I. Biophys. J. 2010; 98: 1751–1761Abstract | Full Text | Full Text PDF | PubMed | Scopus (19)See all References1). The proteases normally circulate in the form of inactive pro-enzymes that can be activated by proteolytic cleavage. Once activated, they cleave and activate other proteases, forming a cascade that powerfully amplifies the initial signal. The cascade begins with so-called “tissue factors,” substances that are exposed to blood when tissues are injured. The end of the cascade is activated factor II (IIa, aka thrombin), which cleaves fibrinogen to form fibrin—the protein polymer that forms the physical body of the clot.Dashkevich et al. (2xThrombin activity propagates in space during blood coagulation as an excitation wave. Dashkevich, N.M., Ovanesov, M.V...., and Ataullakhanov, F.I. Biophys. J. 2012; 103: 2233–2240Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2) in this issue took note of the fact that this highly nonlinear process contains a positive feedback loop because thrombin can also cleave factor XI, higher up in the cascade (3xFeedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis. von dem Borne, P.A., Meijers, J.C., and Bouma, B.N. Blood. 1995; 86: 3035–3042PubMedSee all References3). This suggested that the coagulation system might function as an excitable medium, able to propagate autonomous waves of activation in the same way that a nerve fiber conducts an action potential. To test this hypothesis, they devised an apparatus in which a one-dimensional clotting reaction—instigated by tissue factor at x = 0—could be monitored optically.Thrombin itself is invisible, so the investigators devised a peptide that can be cleaved by thrombin, releasing a fluorophore. To work backward from the fluorescence image to the thrombin distribution, they had to reverse-engineer the reaction-diffusion equation describing this process. Despite the well-known numerical difficulties in inverting such equations, they achieved a usable thrombin image, and demonstrated that the clotting process can, in fact, propagate as an autonomous wave front at the constant—if snail-like—velocity of 20–50 μm/min.To further understand the mechanism, they modified their previous mathematical model of the coagulation cascade (1xTask-oriented modular decomposition of biological networks: trigger mechanism in blood coagulation. Panteleev, M.A., Balandina, A.N...., and Ataullakhanov, F.I. Biophys. J. 2010; 98: 1751–1761Abstract | Full Text | Full Text PDF | PubMed | Scopus (19)See all References1) by adding diffusion terms and the presence of the fluorogenic peptide. This gave a system of 24 partial and two ordinary differential equations, which they numerically integrated. The resulting simulation successfully recapitulated the experimental results, and confirmed that the positive feedback loop from thrombin to factor XI was critical for propagation.To test this conclusion, they repeated the experiment using plasma from patients with hemophilia C, a rare genetic bleeding disorder in which factor XI is absent. As predicted, a sustained, autonomous wave did not form. It has been a long-standing puzzle why hemophilia C causes serious bleeding—particularly from large wounds—when factor XI is only a relatively minor component of the clotting cascade, and one that can be bypassed by other pathways. Based on their experimental and theoretical results, the authors hypothesize that the importance of factor XI lies precisely in its ability to propagate the clotting front far from the injured tissue surface, in order to seal large wounds. They also found that propagation depends on the presence of the cofactor factor VIII, shedding further light on the pathophysiology of the more common Hemophilia A in which that factor is deficient.This work is especially notable for the integration (no pun intended) of theoretical, methodological, and experimental studies. Those of us who do modeling are often tempted simply to demonstrate that a certain mechanism is possible and leave it to others to relate the work to the real world. By intertwining experimental, theoretical, and numerical techniques, these investigators were able to go from a mathematical curiosity to an established mechanism and a proposed solution of a riddle of clinical importance. This is an exemplar of how biophysics should work.

It is well known that the cell membrane is heterogeneous in nature; thereby local and time-stable aggregations of lipids and proteins play a crucial role. The overall protein and lipid distribution within the cell membrane is believed to be strongly influenced by long-range interactions in the membrane plane. These long-range interactions can impossibly directly emerge from typical intermolecular protein-protein interactions such as the H-bonds, the Van der Waals or electrostatic forces, because these forces are too short-ranged (several nanometers).