Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.chemotaxis | single molecule | total internal reflection fluorescence | in vivo microscopy | molecular motor T he bacterial flagellar motor is one of the most complex biological nanomachines (1), ideal for investigating turnover within a multimeric complex. It is the result of the coordinated, sequential expression of about 50 genes (2) producing a structure that spans the cell membrane and rotates an extracellular filament extending several micrometers from the cell surface, at speed of hundreds of hertz. The motor is about is about 45 nm in diameter and is composed of at least 13 different proteins, all in different copy numbers. It is powered by a transmembrane ion flux (1, 3, 4) and consists of a core rotating against a ring of stator proteins (1, 5-7). The C ring, also called the "switch complex," is part of the rotor and localized to the cytoplasmic motor region. The response regulator CheY-P binds one of the C ring components, FliM, causing the rotor to switch rotational direction, thus making FliM the interface with the chemosensory pathway (8-11).Much is known of the static motor structure (5-7), but the dynamics and interactions of its constituents under natural conditions in living cells are poorly understood. Recent results showed that molecules of the stator protein MotB in the flagellar motor, fused to GFP, exchange with a membrane pool of freely diffusing MotB on a time scale of minutes (12)(13)(14). This observation raised the question of whether protein turnover is a general feature of molecular complexes or is a peculiarity of MotB.To ad...

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The Vipp1 protein is essential in cyanobacteria and chloroplasts for the maintenance of photosynthetic function and thylakoid membrane architecture. To investigate its mode of action we generated strains of the cyanobacteria Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 in which Vipp1 was tagged with green fluorescent protein at the C-terminus and expressed from the native chromosomal locus. There was little perturbation of function. Live-cell fluorescence imaging shows dramatic relocalisation of Vipp1 under high light. Under low light, Vipp1 is predominantly dispersed in the cytoplasm with occasional concentrations at the outer periphery of the thylakoid membranes. High light induces Vipp1 coalescence into localised puncta within minutes, with net relocation of Vipp1 to the vicinity of the cytoplasmic membrane and the thylakoid membranes. Pull-downs and mass spectrometry identify an extensive collection of proteins that are directly or indirectly associated with Vipp1 only after high-light exposure. These include not only photosynthetic and stress-related proteins but also RNA-processing, translation and protein assembly factors. This suggests that the Vipp1 puncta could be involved in protein assembly. One possibility is that Vipp1 is involved in the formation of stress-induced localised protein assembly centres, enabling enhanced protein synthesis and delivery to membranes under stress conditions.

Purpose Electrical capacitance tomography (ECT) and electrical resistance tomography (ERT) are promising techniques for multiphase flow measurement due to their high speed, low cost, non-invasive and visualization features. There are two major difficulties in image reconstruction for ECT and ERT: the “soft-field”effect, and the ill-posedness of the inverse problem, which includes two problems: under-determined problem and the solution is not stable, i.e. is very sensitive to measurement errors and noise. This paper aims to summarize and evaluate various reconstruction algorithms which have been studied and developed in the word for many years and to provide reference for further research and application. Design/methodology/approach In the past 10 years, various image reconstruction algorithms have been developed to deal with these problems, including in the field of industrial multi-phase flow measurement and biological medical diagnosis. Findings This paper reviews existing image reconstruction algorithms and the new algorithms proposed by the authors for electrical capacitance tomography and electrical resistance tomography in multi-phase flow measurement and biological medical diagnosis. Originality/value The authors systematically summarize and evaluate various reconstruction algorithms which have been studied and developed in the word for many years and to provide valuable reference for practical applications.

SummaryFluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

Image reconstruction in electrical capacitance tomography requires a solution of an ill-posed inverse problem. This paper applies an accelerated proximal gradient (APG) singular value thresholding algorithm, which is originally proposed for the matrix completion problem, to image two-phase flow. Aiming to improve the image quality, a nuclear norm-based regularization technique is adopted to treat the ill-posedness of the inverse problem, and a simple updating technique is used to update the sensitivity matrix. Both typical and complicated distributions (e.g., "sun-rise" and cross-shape), have been examined based on a 16-electrode configuration. The results showed that the APG algorithm with updated sensitivity matrix could produce higher quality images when compared to the algorithm based on the typical sensitivity matrix. Both simulation and experiment results indicate that the algorithm developed has been able to achieve good quality reconstructed images with relativity fast computation speed for the cases tested in this paper.

Deploying ultra-dense networks that operate on millimeter wave (mmWave) band is a promising way to address the tremendous growth on mobile data traffic. However, one key challenge of ultra-dense mmWave network (UDmmN) is beam management due to the high propagation delay, limited beam coverage as well as numerous beams and users. In this paper, a novel systematic beam control scheme is presented to tackle the beam management problem which is difficult due to the nonconvex objective function. We employ double deep Q-network (DDQN) under a federated learning (FL) framework to address the above optimization problem, and thereby fulfilling adaptive and intelligent beam management in UDmmN. In the proposed beam management scheme based on FL (BMFL), the non-rawdata aggregation can theoretically protect user privacy while reducing handoff cost. Moreover, we propose to adopt a data cleaning technique in the local model training for BMFL, with the aim to further strengthen the privacy protection of users while improving the learning convergence speed. Simulation results demonstrate the performance gain of our proposed scheme.

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