MinD and MinE Interact with Anionic Phospholipids and Regulate Division Plane Formation in Escherichia coli

The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K_d) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K_d for MinD (1.8 μm) in the presence of ATP was smaller than for MinE (12.1 μm) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k_on). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.     

MinDE-Dependent Pole-to-Pole Oscillation of Division Inhibitor MinC in Escherichia coli

By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in Escherichia coli. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell's middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.     

The molecular clockwork of a protein-based circadian oscillator

The circadian clock of the cyanobacterium Synechococcuselongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator’s constituents.     

Distinct brain oscillatory responses for the perception and identification of one’s own body from other’s body

The body recognition process includes complex visual processing, the sensation, perception, and distinction stages of the stimulus. This study examined this process by using the time–frequency analysis of EEG signals and analyzed the obtained data by using the event-related oscillations method. This study aimed to examine the oscillatory brain responses and distinguish one’s own body from other’s body. In the present study, 17 young adults were included and the EEGs were recorded with 32 electrodes placed in different locations. Event-related power spectrum and phase-locking analyzes were performed. ITC and ERSP data were analyzed using 2 (condition) × 11 (location) × 2 (hemisphere) ANOVA Design. As we observed a prolonged response in the theta band in the grand averages, we included the time variable in the overall model. As a result, we found that the phase-locking and the event-related power spectrum of the theta response in recognizing one’s own body were higher when compared to the phase-locking and the event-related power spectrum of the theta response in recognizing others’ body (p < 0.05). When the time variable was included, the early theta response was more phase-locked and had a higher power spectrum compared to the late theta response (p < 0.05). As a result of the power spectrum analysis, the condition × hemisphere interaction effect in the beta band was higher in the left hemisphere regarding increased responses in recognizing one’s own body (p < 0.05). As a result of ITC, the main effect of the condition was higher in the recognition of the stimulus of one’s own body (p < 0.05). Finally, the theta oscillator response stood out in distinguishing one’s own body from other’s body. Similarly, the power spectrum in the beta response was higher in the left hemisphere, and this finding is consistent with the literature.     

A Canonical Circuit for Generating Phase-Amplitude Coupling

‘Phase amplitude coupling’ (PAC) in oscillatory neural activity describes a phenomenon whereby the amplitude of higher frequency activity is modulated by the phase of lower frequency activity. Such coupled oscillatory activity – also referred to as ‘cross-frequency coupling’ or ‘nested rhythms’ – has been shown to occur in a number of brain regions and at behaviorally relevant time points during cognitive tasks; this suggests functional relevance, but the circuit mechanisms of PAC generation remain unclear. In this paper we present a model of a canonical circuit for generating PAC activity, showing how interconnected excitatory and inhibitory neural populations can be periodically shifted in to and out of oscillatory firing patterns by afferent drive, hence generating higher frequency oscillations phase-locked to a lower frequency, oscillating input signal. Since many brain regions contain mutually connected excitatory-inhibitory populations receiving oscillatory input, the simplicity of the mechanism generating PAC in such networks may explain the ubiquity of PAC across diverse neural systems and behaviors. Analytic treatment of this circuit as a nonlinear dynamical system demonstrates how connection strengths and inputs to the populations can be varied in order to change the extent and nature of PAC activity, importantly which phase of the lower frequency rhythm the higher frequency activity is locked to. Consequently, this model can inform attempts to associate distinct types of PAC with different network topologies and physiologies in real data.     

Gateway-Based Destination Vectors for Functional Analyses of Bacterial ORFeomes: Application to the Min System in Brucella abortus

Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscillating localization of MinD in Brucella abortus.     

Enhancements to the Rosetta Energy Function Enable Improved Identification of Small Molecules that Inhibit Protein-Protein Interactions

Protein-protein interactions are among today’s most exciting and promising targets for therapeutic intervention. To date, identifying small-molecules that selectively disrupt these interactions has proven particularly challenging for virtual screening tools, since these have typically been optimized to perform well on more “traditional” drug discovery targets. Here, we test the performance of the Rosetta energy function for identifying compounds that inhibit protein interactions, when these active compounds have been hidden amongst pools of “decoys.” Through this virtual screening benchmark, we gauge the effect of two recent enhancements to the functional form of the Rosetta energy function: the new “Talaris” update and the “pwSHO” solvation model. Finally, we conclude by developing and validating a new weight set that maximizes Rosetta’s ability to pick out the active compounds in this test set. Looking collectively over the course of these enhancements, we find a marked improvement in Rosetta’s ability to identify small-molecule inhibitors of protein-protein interactions.     

Gamma Oscillations as a Neural Signature of Shifting Times in Narrative Language

Verbs and other temporal expressions allow speakers to specify the location of events in time, as well as to move back and forth in time, shifting in a narrative between past, present and future. The referential flexibility of temporal expressions is well understood in linguistics but its neurocognitive bases remain unknown. We aimed at obtaining a neural signature of shifting times in narrative language. We recorded and analyzed event-related brain potentials (ERPs) and oscillatory responses to the adverb ‘now’ and to the second main verb in Punctual (‘An hour ago the boy stole a candy and now he peeled the fruit’) and Iterative (‘The entire afternoon the boy stole candy and now he peeled the fruit’) contexts. ‘An hour ago’ introduces a time frame that lies entirely in the past, ‘now’ shifts the narrative to the present, and ‘peeled’ shifts it back to the past. These two referential shifts in Punctual contexts are expected to leave very similar traces on neural responses. In contrast, ‘The entire afternoon’ specifies a time frame that may encompass past, present and future, such that both ‘now’ and ‘peeled’ are consistent with it. Here, no time shift is required. We found no difference in ERPs between Punctual and Iterative contexts either at ‘now’ or at the second verb. However, reference shifts modulated oscillatory signals. ‘Now’ and the second verb in Punctual contexts resulted in similar responses: an increase in gamma power with a left-anterior distribution. Gamma bursts were absent in Iterative contexts. We propose that gamma oscillations here reflect the binding of temporal variables to the values allowed by constraints introduced by temporal expressions in discourse.     

A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum

MinD is a key component of an oscillatory system that spatially regulates cell division in Escherichia coli. It is a peripheral membrane ATPase that recruits MinC and oscillates between the two halves of the cell in a MinE dependent manner. In vitro MinD binds to phospholipid vesicles in an ATP-dependent manner and is released through MinE-stimulated ATP hydrolysis. In this study we examined the function of the conserved C-terminus of MinD. Short truncations of three and ten amino acids dramatically decreased the ability of MinD to localize to the membrane and spatially regulate division. These truncations bound MinC but were deficient in targeting MinC to the septum. In vitro they dimerized, but were deficient in binding to phospholipid vesicles and undergoing MinE stimulation. We suggest a model in which the ATP-dependent dimerization of MinD affects the conformation of the C-terminal region, a potential amphipathic helix, triggering membrane binding.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Robust Min‐system oscillation in the presence of internal photosynthetic membranes in cyanobacteria

The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ‐ring formation and represents one of the best‐understood examples of emergent protein self‐organization in nature. The oscillatory patterns of the Min‐system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self‐organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, it was shown that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole‐to‐pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction‐diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Interestingly, in addition to oscillating, MinC exhibits a midcell localization dependent on MinD and the DivIVA‐like protein Cdv3, indicating that two distinct pools of MinC are coordinated in S. elongatus. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min‐system function in bacteria and organelles with internal membrane systems.     

Evaluation of the Oscillatory Interference Model of Grid Cell Firing through Analysis and Measured Period Variance of Some Biological Oscillators

Models of the hexagonally arrayed spatial activity pattern of grid cell firing in the literature generally fall into two main categories: continuous attractor models or oscillatory interference models. Burak and Fiete (2009, PLoS Comput Biol) recently examined noise in two continuous attractor models, but did not consider oscillatory interference models in detail. Here we analyze an oscillatory interference model to examine the effects of noise on its stability and spatial firing properties. We show analytically that the square of the drift in encoded position due to noise is proportional to time and inversely proportional to the number of oscillators. We also show there is a relatively fixed breakdown point, independent of many parameters of the model, past which noise overwhelms the spatial signal. Based on this result, we show that a pair of oscillators are expected to maintain a stable grid for approximately t = 5µ ³ /(4πσ) ² seconds where µ is the mean period of an oscillator in seconds and σ² its variance in seconds². We apply this criterion to recordings of individual persistent spiking neurons in postsubiculum (dorsal presubiculum) and layers III and V of entorhinal cortex, to subthreshold membrane potential oscillation recordings in layer II stellate cells of medial entorhinal cortex and to values from the literature regarding medial septum theta bursting cells. All oscillators examined have expected stability times far below those seen in experimental recordings of grid cells, suggesting the examined biological oscillators are unfit as a substrate for current implementations of oscillatory interference models. However, oscillatory interference models can tolerate small amounts of noise, suggesting the utility of circuit level effects which might reduce oscillator variability. Further implications for grid cell models are discussed.     

Lack of Dietary Polyunsaturated Fatty Acids Causes Synapse Dysfunction in the Drosophila Visual System

Polyunsaturated fatty acids (PUFAs) are essential nutrients for animals and necessary for the normal functioning of the nervous system. A lack of PUFAs can result from the consumption of a deficient diet or genetic factors, which impact PUFA uptake and metabolism. Both can cause synaptic dysfunction, which is associated with numerous disorders. However, there is a knowledge gap linking these neuronal dysfunctions and their underlying molecular mechanisms. Because of its genetic manipulability and its easy, fast, and cheap breeding, Drosophila melanogaster has emerged as an excellent model organism for genetic screens, helping to identify the genetic bases of such events. As a first step towards the understanding of PUFA implications in Drosophila synaptic physiology we designed a breeding medium containing only very low amounts of PUFAs. We then used the fly’s visual system, a well-established model for studying signal transmission and neurological disorders, to measure the effects of a PUFA deficiency on synaptic function. Using both visual performance and eye electrophysiology, we found that PUFA deficiency strongly affected synaptic transmission in the fly’s visual system. These defects were rescued by diets containing omega-3 or omega-6 PUFAs alone or in combination. In summary, manipulating PUFA contents in the fly’s diet was powerful to investigate the role of these nutrients on the fly´s visual synaptic function. This study aims at showing how the first visual synapse of Drosophila can serve as a simple model to study the effects of PUFAs on synapse function. A similar approach could be further used to screen for genetic factors underlying the molecular mechanisms of synaptic dysfunctions associated with altered PUFA levels.     

The series elastic shock absorber: tendon elasticity modulates energy dissipation by muscle during burst deceleration

During downhill running, manoeuvring, negotiation of obstacles and landings from a jump, mechanical energy is dissipated via active lengthening of limb muscles. Tendon compliance provides a ‘shock-absorber’ mechanism that rapidly absorbs mechanical energy and releases it more slowly as the recoil of the tendon does work to stretch muscle fascicles. By lowering the rate of muscular energy dissipation, tendon compliance likely reduces the risk of muscle injury that can result from rapid and forceful muscle lengthening. Here, we examine how muscle–tendon mechanics are modulated in response to changes in demand for energy dissipation. We measured lateral gastrocnemius (LG) muscle activity, force and fascicle length, as well as leg joint kinematics and ground-reaction force, as turkeys performed drop-landings from three heights (0.5–1.5 m centre-of-mass elevation). Negative work by the LG muscle–tendon unit during landing increased with drop height, mainly owing to greater muscle recruitment and force as drop height increased. Although muscle strain did not increase with landing height, ankle flexion increased owing to increased tendon strain at higher muscle forces. Measurements of the length–tension relationship of the muscle indicated that the muscle reached peak force at shorter and likely safer operating lengths as drop height increased. Our results indicate that tendon compliance is important to the modulation of energy dissipation by active muscle with changes in demand and may provide a mechanism for rapid adjustment of function during deceleration tasks of unpredictable intensity.     

Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood

Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne’s Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne’s muscular dystrophy. Both in JDM and Duchenne’s muscular dystrophy the proportion of FOXP3⁺ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.