The mechanism of activation of MEK1 by B-Raf and KSR1.

MEK1 interactions with B-Raf and KSR1 are key steps in Ras/Raf/MEK/ERK signaling. Despite this, vital mechanistic details of how these execute signal transduction are still enigmatic. Among these is why, despite B-Raf and KSR1 kinase domains similarity, the B-Raf/MEK1 and KSR1/MEK1 complexes have distinct contributions to MEK1 activation, and broadly, what is KSR1's role. Our molecular dynamics simulations clarify these still unresolved ambiguities. Our results reveal that the proline-rich (P-rich) loop of MEK1 plays a decisive role in MEK1 activation loop (A-loop) phosphorylation. In the inactive B-Raf/MEK1 heterodimer, the collapsed A-loop of B-Raf interacts with the P-rich loop and A-loop of MEK1, minimizing MEK1 A-loop fluctuation and preventing it from phosphorylation. In the active B-Raf/MEK1 heterodimer, the P-rich loop moves in concert with the A-loop of B-Raf as it extends. This reduces the number of residues interacting with MEK1 A-loop, allowing increased A-loop fluctuation, and bringing Ser222 closer to ATP for phosphorylation. B-Raf αG-helix Arg662 promotes MEK1 activation by orienting Ser218 towards ATP. In KSR1/MEK1, the KSR1 αG-helix has Ala826 in place of B-Raf Arg662. This difference results in much fewer interactions between KSR1 αG-helix and MEK1 A-loop, thus a more flexible A-loop. We postulate that if KSR1 were to adopt an active configuration with an extended A-loop as seen in other protein kinases, then the MEK1 P-rich loop would extend in a similar manner, as seen in the active B-Raf/MEK1 heterodimer. This would result in highly flexible MEK1 A-loop, and KSR1 functioning as an active, B-Raf-like, kinase.

The structural basis of BCR-ABL recruitment of GRB2 in chronic myelogenous leukemia.

BCR-ABL drives chronic myeloid leukemia (CML). BCR binding to GRB2 transduces signaling via the Ras/MAPK pathway. Despite considerable data confirming the binding, molecular-level understanding of exactly how the two proteins interact, and, especially, what are the determinants of the specificity of the SH2GRB2 domain-phosphorylated BCR (pBCR) recognition are still open questions. Yet, this is vastly important for understanding binding selectivity, and for predicting the phosphorylated receptors, or peptides, that are likely to bind. Here, we uncover these determinants and ascertain to what extent they relate to the affinity of the interaction. Toward this end, we modeled the complexes of the pBCR and SH2GRB2 and other pY/Y-peptide-SH2 complexes and compared their specificity and affinity. We observed that pBCR's 176FpYVNV180 motif is favorable and specific to SH2GRB2, similar to pEGFR, but not other complexes. SH2GRB2 contains two binding pockets: pY-binding recognition pocket triggers binding, and the specificity pocket whose interaction is governed by N179 in pBCR and W121 in SH2GRB2. Our proposed motif with optimal affinity to SH2GRB2 is E/D-pY-E/V-N-I/L. Collectively, we provide the structural basis of BCR-ABL recruitment of GRB2, outline its specificity hallmarks, and delineate a blueprint for prediction of BCR-binding scaffolds and for therapeutic peptide design.

Mechanism of activation and the rewired network: New drug design concepts.

Precision oncology benefits from effective early phase drug discovery decisions. Recently, drugging inactive protein conformations has shown impressive successes, raising the cardinal questions of which targets can profit and what are the principles of the active/inactive protein pharmacology. Cancer driver mutations have been established to mimic the protein activation mechanism. We suggest that the decision whether to target an inactive (or active) conformation should largely rest on the protein mechanism of activation. We next discuss the recent identification of double (multiple) same-allele driver mutations and their impact on cell proliferation and suggest that like single driver mutations, double drivers also mimic the mechanism of activation. We further suggest that the structural perturbations of double (multiple) in cis mutations may reveal new surfaces/pockets for drug design. Finally, we underscore the preeminent role of the cellular network which is deregulated in cancer. Our structure-based review and outlook updates the traditional Mechanism of Action, informs decisions, and calls attention to the intrinsic activation mechanism of the target protein and the rewired tumor-specific network, ushering innovative considerations in precision medicine.

Asymmetries between achromatic and chromatic extraction of 3D motion signals.

Motion in depth (MID) can be cued by high-resolution changes in binocular disparity over time (CD), and low-resolution interocular velocity differences (IOVD). Computational differences between these two mechanisms suggest that they may be implemented in visual pathways with different spatial and temporal resolutions. Here, we used fMRI to examine how achromatic and S-cone signals contribute to human MID perception. Both CD and IOVD stimuli evoked responses in a widespread network that included early visual areas, parts of the dorsal and ventral streams, and motion-selective area hMT+. Crucially, however, we measured an interaction between MID type and chromaticity. fMRI CD responses were largely driven by achromatic stimuli, but IOVD responses were better driven by isoluminant S-cone inputs. In our psychophysical experiments, when S-cone and achromatic stimuli were matched for perceived contrast, participants were equally sensitive to the MID in achromatic and S-cone IOVD stimuli. In comparison, they were relatively insensitive to S-cone CD. These findings provide evidence that MID mechanisms asymmetrically draw on information in precortical pathways. An early opponent motion signal optimally conveyed by the S-cone pathway may provide a substantial contribution to the IOVD mechanism.

Clustering and Phase Separation in Mixtures of Dipolar and Active Particles in an External Field.

Directing the assembly of colloidal particles through the use of external electric or magnetic fields shows promise for the creation of reconfigurable materials. Self-propelled particles can also be used to dynamically drive colloidal systems to nonequilibrium steady states. We investigate colloidal systems that combine both of these methods of directed assembly, simulating mixtures of passive dipolar colloids and active soft spheres in an external magnetic field using Brownian dynamics in two dimensions. In these systems, the dipolar particles align in the direction of the external field, but the active particles are unaffected by the field. The phase behaviors exhibited included a percolated dipolar network, dipolar string-fluid, isotropic fluid, and phase-separated state. We find that the external field allows the dipolar particles to form a percolated network more easily compared to when no external field is present. Additionally, the mixture phase separates at lower active particle velocity in an external field than when no field is present. Our results suggest that combining multiple methods of directing colloidal assembly could lead to new pathways to fabricate reconfigurable materials.

Clustering and phase separation in mixtures of dipolar and active particles.

The self-assembly of colloidal particles in dynamic environments has become an important field of study because of potential applications in fabricating out-of-equilibrium materials. We investigate the phase behavior of mixtures of passive dipolar colloids and active soft spheres using Brownian dynamics simulations in two dimensions. The phase behaviors exhibited include dipolar percolated network, dipolar string-fluid, isotropic fluid, and a phase-separated state. We find that the clustering of dipolar colloids is enhanced in the presence of slow-moving active particles compared to the clustering of dipolar particles mixed with passive particles. When the active particle motility is high, the chains of dipolar particles are either broken into short chains or pushed into dense clusters. Motility-induced phase separation into dense and dilute phases is also present. The area fraction of particles in the dilute phase increases as the fraction of active particles in the system decreases, while the area fraction of particles in the dense phase remains constant. Our findings are relevant to the development of reconfigurable self-assembled materials.

Phase diagrams of mixtures of dipolar rods and discs.

Self-assembly of binary mixtures that contain anisotropic, interacting colloidal particles have been proposed as a way to create new, multi-functional materials. We simulate binary mixtures of dipolar rods and dipolar discs in two-dimensions using discontinuous molecular dynamics to determine how the assembled structures of these mixtures differ from those seen in single component systems. Two different binary mixtures are investigated: a mixture of an equal number of dipolar rods and dipolar discs ("equal number"), and a mixture where the area fraction of dipolar rods is equal to the area fraction of dipolar discs ("equal area"). Phase boundaries between fluid, string-fluid, and "gel" phases are calculated and compared to the phase boundaries of the pure components. Looking deeper at the underlying structure of the mixture reveals a complex interplay between the rods and discs and the formation of states where the two components are in different phases. The mixtures exhibit phases where both rods and discs are in the fluid phase, where rods form a string-fluid while discs remain in the fluid phase, a rod string-fluid coexisting with a disc string-fluid, a "gel" that consists primarily of rods while the discs form either a fluid or string-fluid phase, and a "gel" that contains both rods and discs. Our results give insight into the general assembly pathway of binary mixtures, and how complex aggregates can be created by varying the mixture composition, strength of interaction between the two components, and the temperature. By manipulating the properties of one of the components it should be possible to fabricate bifunctional, thermally responsive self-assembled materials.

Directional Limits on Motion Transparency Assessed Through Colour-Motion Binding.

Motion-defined transparency is the perception of two or more distinct moving surfaces at the same retinal location. We explored the limits of motion transparency using superimposed surfaces of randomly positioned dots defined by differences in motion direction and colour. In one experiment, dots were red or green and we varied the proportion of dots of a single colour that moved in a single direction ('colour-motion coherence') and measured the threshold direction difference for discriminating between two directions. When colour-motion coherences were high (e.g., 90% of red dots moving in one direction), a smaller direction difference was required to correctly bind colour with direction than at low coherences. In another experiment, we varied the direction difference between the surfaces and measured the threshold colour-motion coherence required to discriminate between them. Generally, colour-motion coherence thresholds decreased with increasing direction differences, stabilising at direction differences around 45°. Different stimulus durations were compared, and thresholds were higher at the shortest (150 ms) compared with the longest (1,000 ms) duration. These results highlight different yet interrelated aspects of the task and the fundamental limits of the mechanisms involved: the resolution of narrowly separated directions in motion processing and the local sampling of dot colours from each surface.

Probing the Characteristics of Colour-Motion Binding and Its Dependence on Persistent Surface Segregation.

Identifying the spatial and temporal characteristics of visual feature binding is a remaining challenge in the science of perception. Within the feature-binding literature, disparate findings have suggested the existence of more than one feature-binding mechanism with differing temporal resolutions. For example, one surprising result is that temporal alternations between two different feature pairings of colour and motion (e.g., orange dots moving left with blue dots moving right) support accurate conjunction discrimination at alternation frequencies of around 10 Hz and greater. However, at lower alternation frequencies around 5 Hz, conjunction discrimination falls to chance. To further investigate this effect, we present two experiments that probe the stimulus characteristics that facilitate or impede feature binding. Using novel manipulations of random dot kinematograms, we identify that facilitating surface representations through temporal integration can enable accurate conjunction discrimination at both intermediate and high alternation frequencies. We also offer a neurally plausible evidence accumulator model to describe these results, removing the need to suggest multiple binding mechanisms acting at different timescales. In effect, we propose a single, flexible binding process, whereby the relatively low temporal resolution for binding features can be circumvented by extracting them from rapidly formed and persistent surface representations.

Orientation anisotropies in human primary visual cortex depend on contrast.

Orientation processing in visual cortex appears matched to the environment, such that larger neural populations are tuned to cardinal (horizontal/vertical) than oblique orientations. This may be manifested perceptually as a cardinal bias: poorer sensitivity to oblique compared to cardinal orientations (the "oblique effect"). However, a growing body of psychophysical data reveals the opposite pattern of anisotropy: a bias towards the oblique over the cardinal orientations (the "horizontal effect"), something matched by recent functional magnetic resonance imaging (fMRI) studies that have found an increased response to the oblique over the cardinal orientations in early visual cortex. This may reveal the operation of an efficient coding strategy optimised to the diet of orientations encountered during natural viewing. From consideration of coding efficiency, it might be expected that the anisotropies would change as the quality/strength of the oriented stimulus changes. In two experiments, fMRI response modulations were measured in retinotopically-defined human early visual cortex as a function of the contrast and orientation of sinusoidal gratings. Both experiments revealed a marked change in the V1 response from a cardinal (vertical) bias at low contrast to an oblique bias at high contrast. In Experiment 2, this was also apparent in areas V2 and V3. On average, there was no systematic "radial bias" (a preference for orientations aligned with the visual field meridian) in V1, although it was present in some individual subjects. The change in orientation anisotropies with contrast is consistent with an adaptive stimulus coding strategy in cortex that shifts according to the strength of the sensory inputs.

The basis of orientation decoding in human primary visual cortex: fine- or coarse-scale biases?

Orientation signals in human primary visual cortex (V1) can be reliably decoded from the multivariate pattern of activity as measured with functional magnetic resonance imaging (fMRI). The precise underlying source of these decoded signals (whether by orientation biases at a fine or coarse scale in cortex) remains a matter of some controversy, however. Freeman and colleagues (J Neurosci 33: 19695-19703, 2013) recently showed that the accuracy of decoding of spiral patterns in V1 can be predicted by a voxel's preferred spatial position (the population receptive field) and its coarse orientation preference, suggesting that coarse-scale biases are sufficient for orientation decoding. Whether they are also necessary for decoding remains an open question, and one with implications for the broader interpretation of multivariate decoding results in fMRI studies.

Transparent surface segregation enables visual feature binding in rapidly alternating displays.

Visual feature binding-the mechanism by which our typically coherent and unified perceptual experience arises from distributed neural representations-is the source of much intrigue in the neuroscience of perception. Surprisingly, feature binding can occur in rapidly alternating displays of color-orientation combinations (e.g., rightward-orange, leftward-blue). However, we found that when the angular separation between orientations is reduced, binding is selectively impaired at temporal alternation frequencies around 5 Hz. To isolate the mechanisms involved, we devised a novel display in which color-orientation conjunction information was distributed temporally over two checkered stimuli and was perceptually discriminable only within an intermediate range of temporal frequencies (7.5-15 Hz). We propose that accurate color-orientation judgments at frequencies exceeding 5 Hz depend on the rapid formation of persistent surface representations that can be accessed by binding mechanisms, circumventing the latter's relatively low temporal resolution.

Motion-defined surface segregation in human visual cortex.

Surface segregation provides an efficient way to parse the visual scene for perceptual analysis. Here, we investigated the segregation of a bivectorial motion display into transparent surfaces through a psychophysical task and fMRI. We found that perceptual transparency correlated with neural activity in the early areas of the visual cortex, suggesting these areas may be involved in the segregation of motion-defined surfaces. Two oppositely rotating, uniquely colored random dot kinematograms (RDKs) were presented either sequentially or in a spatially interleaved manner, displayed at varying alternation frequencies. Participants reported the color and rotation direction pairing of the RDKs in the psychophysical task. The spatially interleaved display generated the percept of motion transparency across the range of frequencies tested, yielding ceiling task performance. At high alternation frequencies, performance on the sequential display also approached ceiling, indicative of perceived transparency. However, transparency broke down in lower alternation frequency sequential displays, producing performance close to chance. A corresponding pattern mirroring the psychophysical data was also evident in univariate and multivariate analyses of the fMRI BOLD activity in visual cortical areas V1, V2, V3, V3AB, hV4, and V5/MT+. Using gray RDKs, we found significant presentation by frequency interactions in most areas; differences in BOLD signal between presentation types were significant only at the lower alternation frequency. Multivariate pattern classification was similarly unable to discriminate between presentation types at the higher frequency. This study provides evidence that early visual cortex may code for motion-defined surface segregation, which in turn may enable perceptual transparency.

Determinants of motion response anisotropies in human early visual cortex: the role of configuration and eccentricity.

Anisotropies in the cortical representation of various stimulus parameters can reveal the fundamental mechanisms by which sensory properties are analysed and coded by the brain. One example is the preference for motion radial to the point of fixation (i.e. centripetal or centrifugal) exhibited in mammalian visual cortex. In two experiments, this study used functional magnetic resonance imaging (fMRI) to explore the determinants of these radial biases for motion in functionally-defined areas of human early visual cortex, and in particular their dependence upon eccentricity which has been indicated in recent reports. In one experiment, the cortical response to wide-field random dot kinematograms forming 16 different complex motion patterns (including centrifugal, centripetal, rotational and spiral motion) was measured. The response was analysed according to preferred eccentricity within four different eccentricity ranges. Response anisotropies were characterised by enhanced activity for centripetal or centrifugal patterns that changed systematically with eccentricity in visual areas V1-V3 and hV4 (but not V3A/B or V5/MT+). Responses evolved from a preference for centrifugal over centripetal patterns close to the fovea, to a preference for centripetal over centrifugal at the most peripheral region stimulated, in agreement with previous work. These effects were strongest in V2 and V3. In a second experiment, the stimuli were restricted to within narrow annuli either close to the fovea (0.75-1.88°) or further in the periphery (4.82-6.28°), in a way that preserved the local motion information available in the first experiment. In this configuration a preference for radial motion (centripetal or centrifugal) persisted but the dependence upon eccentricity disappeared. Again this was clearest in V2 and V3. A novel interpretation of the dependence upon eccentricity of motion anisotropies in early visual cortex is offered that takes into account the spatiotemporal "predictability" of the moving pattern. Such stimulus predictability, and its relationship to models of predictive coding, has found considerable support in recent years in accounting for a number of other perceptual and neural phenomena.

Conversion efficiency of skutterudite-based thermoelectric modules.

Presently, the only commercially available power generating thermoelectric (TE) modules are based on bismuth telluride (Bi2Te3) alloys and are limited to a hot side temperature of 250 °C due to the melting point of the solder interconnects and/or generally poor power generation performance above this point. For the purposes of demonstrating a TE generator or TEG with higher temperature capability, we selected skutterudite based materials to carry forward with module fabrication because these materials have adequate TE performance and are mechanically robust. We have previously reported the electrical power output for a 32 couple skutterudite TE module, a module that is type identical to ones used in a high temperature capable TEG prototype. The purpose of this previous work was to establish the expected power output of the modules as a function of varying hot and cold side temperatures. Recent upgrades to the TE module measurement system built at the Fraunhofer Institute for Physical Measurement Techniques allow for the assessment of not only the power output, as previously described, but also the thermal to electrical energy conversion efficiency. Here we report the power output and conversion efficiency of a 32 couple, high temperature skutterudite module at varying applied loading pressures and with different interface materials between the module and the heat source and sink of the test system. We demonstrate a 7% conversion efficiency at the module level when a temperature difference of 460 °C is established. Extrapolated values indicate that 7.5% is achievable when proper thermal interfaces and loading pressures are used.

Determinants of the direction illusion: motion speed and dichoptic presentation interact to reveal systematic individual differences in sign.

When two fields of dots with different directions of movement are presented in tandem, the perceived direction of one is biased by the presence of the other. Although this ''direction illusion'' typically involves repulsion, with an exaggeration of the perceived angular difference in direction between the dot fields, attraction effects, where the perceived difference is reduced, have also been found under certain presentation conditions. Earlier literature has been inconsistent, and there is debate surrounding the nature of the interactions that facilitate the direction illusion, as well as whether they occur at a local or global stage of the motion processing hierarchy. Here we measured the operating characteristics of the direction illusion by parametrically varying inducer contrast and coherence while examining the effects of stimulus speed and dichoptic presentation. It was found that the magnitude and sign of the direction illusion differed substantially from earlier research. Furthermore, there appeared to be significant interindividual variability, with dichoptic presentation producing an attractive rather than repulsive direction illusion in some participants.

Efficacy and specificity of melanopsin reporters for retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are specialized retinal output neurons that mediate behavioral, neuroendocrine, and developmental responses to environmental light. There are diverse molecular strategies for marking ipRGCs, especially in mice, making them among the best characterized retinal ganglion cells (RGCs). With the development of more sensitive reporters, new subtypes of ipRGCs have emerged. We therefore tested high-sensitivity reporter systems to see whether we could reveal yet more. Substantial confusion remains about which of the available methods, if any, label all and only ipRGCs. Here, we compared many different methods for labeling of ipRGCs, including anti-melanopsin immunofluorescence, Opn4-GFP BAC transgenic mice, and Opn4cre mice crossed with three different Cre-specific reporters (Z/EG, Ai9, and Ai14) or injected with Cre-dependent (DIO) AAV2. We show that Opn4cre mice, when crossed with sensitive Cre-reporter mice, label numerous ganglion cell types that lack intrinsic photosensitivity. Though other methods label ipRGCs specifically, they do not label the entire population of ipRGCs. We conclude that no existing method labels all and only ipRGCs. We assess the appropriateness of each reporter for particular applications and integrate findings across reporters to estimate that the overall abundance of ipRGCs among mouse RGCs may approach 11%.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Human cortical and behavioral sensitivity to patterns of complex motion at eccentricity.

Complex patterns of image motion (contracting, expanding, rotating, and spiraling fields) are important in the coordination of visually guided behaviors. Whereas specialized detectors in monkey visual cortex show selectivity for particular patterns of complex motion, their representation in human visual cortex remains unclear. In the present study, functional magnetic resonance imaging (fMRI) was used to investigate the sensitivity of functionally defined regions of human visual cortex to parametrically modulated complex motion trajectories, coupled with complementary psychophysical testing. A unique stimulus design made it possible to disambiguate the neural responses and psychophysical sensitivity to complex motions per se from the distribution of local motions relative to the fovea, which are known to enhance cortical activity when presented radial to fixation. This involved presenting several small, separate motion fields in the periphery in a manner that distinguished them from global optic flow patterns. The patterns were morphed through complex motion space in a systematic time-locked fashion when presented in the scanner. Anisotropies were observed in the fMRI signal, marked by an enhanced response to expanding vs. contracting fields, even in early visual cortex. Anisotropies in the psychophysical sensitivity measures followed a similar pattern that was correlated with activity in areas hV4, V5/MT, and MST. This represents the first systematic examination of complex motion perception at both a behavioral and neural level in human observers. The characteristic processing anisotropy revealed in both data sets can inform models of complex motion processing, particularly with respect to computations performed in early visual cortex.

Colour misbinding during motion rivalry.

When two dissimilar colours are displayed to the two eyes at overlapping retinal locations, binocular rivalry typically results: a fluctuating struggle for perceptual dominance of each eye's stimulus. We found instead that isoluminant counter-rotating patterns consisting of coloured and achromatic portions can promote an illusory colour 'misbinding', where the colours from both eyes were perceived within a single rotating pattern. The achromatic portion of one rotating pattern thus appeared to take on the colour of the other, oppositely rotating pattern. The results suggest that the neural mechanisms of colour binding can operate even while representations of the same patterns' motions are undergoing rivalry, and support the idea that rivalry can occur in isolation within the motion system.