Clock proteins regulate spatiotemporal organization of clock genes to control circadian rhythms

Circadian clocks regulate ∼24-h oscillations in gene expression, behavior, and physiology. While the genetic and molecular mechanisms of circadian rhythms are well characterized, what remains poorly understood are the intracellular dynamics of circadian clock components and how they affect circadian rhythms. Here, we elucidate how spatiotemporal organization and dynamics of core clock proteins and genes affect circadian rhythms in Drosophila clock neurons. Using high-resolution imaging and DNA-fluorescence in situ hybridization techniques, we demonstrate that Drosophila clock proteins (PERIOD and CLOCK) are organized into a few discrete foci at the nuclear envelope during the circadian repression phase and play an important role in the subnuclear localization of core clock genes to control circadian rhythms. Specifically, we show that core clock genes, period and timeless, are positioned close to the nuclear periphery by the PERIOD protein specifically during the repression phase, suggesting that subnuclear localization of core clock genes might play a key role in their rhythmic gene expression. Finally, we show that loss of Lamin B receptor, a nuclear envelope protein, leads to disruption of PER foci and per gene peripheral localization and results in circadian rhythm defects. These results demonstrate that clock proteins play a hitherto unexpected role in the subnuclear reorganization of core clock genes to control circadian rhythms, revealing how clocks function at the subcellular level. Our results further suggest that clock protein foci might regulate dynamic clustering and spatial reorganization of clock-regulated genes over the repression phase to control circadian rhythms in behavior and physiology.

Myosin V Regulates Spatial Localization of Different Forms of Neurotransmitter Release in Central Synapses

Synaptic active zone (AZ) contains multiple specialized release sites for vesicle fusion. The utilization of release sites is regulated to determine spatiotemporal organization of the two main forms of synchronous release, uni-vesicular (UVR) and multi-vesicular (MVR). We previously found that the vesicle-associated molecular motor myosin V regulates temporal utilization of release sites by controlling vesicle anchoring at release sites in an activity-dependent manner. Here we show that acute inhibition of myosin V shifts preferential location of vesicle docking away from AZ center toward periphery, and results in a corresponding spatial shift in utilization of release sites during UVR. Similarly, inhibition of myosin V also reduces preferential utilization of central release sites during MVR, leading to more spatially distributed and temporally uniform MVR that occurs farther away from the AZ center. Using a modeling approach, we provide a conceptual framework that unites spatial and temporal functions of myosin V in vesicle release by controlling the gradient of release site release probability across the AZ, which in turn determines the spatiotemporal organization of both UVR and MVR. Thus myosin V regulates both temporal and spatial utilization of release sites during two main forms of synchronous release.

Cold Adaptation Leads To Repolarization Gradients Development In The Rainbow Trout Heart

ABSTRACTIntroductionThermal adaptation in fish is accompanied by morphological and electrophysiological changes in the myocardium. Little is known regarding changes of spatiotemporal organization of ventricular excitation and repolarization processes with acclimatization. We aimed to evaluate transmural and apicobasal heterogeneity of depolarization and repolarization characteristics in the in-situ heart of rainbow trout in seasonal acclimatization.MethodsThe experiments were done in the summer-acclimatized (SA, 18°C, n=8) and winter-acclimatized (WA, 3°C, n=8) rainbow trout. 24 unipolar electrograms were recorded with plunge needle electrodes (eight lead terminals each) impaled into the ventricular wall. Activation time (AT), end of repolarization time (RT), and activation-repolarization interval (ARI, a surrogate for action potential duration) were determined as dV/dt min during QRS-complex, dV/dt max during T-wave, and RT-AT difference, respectively.ResultsThe SA fish demonstrated relatively flat apicobasal and transmural AT and especially ARI profiles. In the WA animals, ATs and ARIs were longer as compared to SA animals (p≤0.001), ARIs were shorter in the compact layer than in the spongy layer (p≤0.050), and within the compact layer, the apical region had shorter ATs and longer ARIs as compared to the basal region (p≤0.050). In multiple linear regression analysis, ARI duration was associated with cardiac cycle duration and AT in SA and WA animals. The WA animals demonstrated additionally an independent association of ARIs with spatial localization across the ventricle.ConclusionAdaptation to cold conditions in rainbow trout was associated with a spatial ventricular remodeling leading to the development of repolarization gradients typically observed in mammalian myocardium.SUMMARY STATEMENTThe study gives an example of thermal adaptation in fish realized at the level of spatiotemporal organization of myocardial depolarization and repolarization.

Lag structure of BOLD signals within white matter in resting-state fMRI

Previous studies have demonstrated that BOLD signals in gray matter in resting-state functional MRI (RSfMRI) have variable time lags. We investigated the corresponding variations of signal latencies in white matter within 1393 subjects (both sexes included) from the Brain Genomics Superstruct Project. We divided the dataset into ten equal groups to study both the patterns and reproducibility of latency estimates within white matter. We constructed time delay matrices by computing cross-correlation functions between voxel pairs. Projections of voxel latencies were highly correlated (average Pearson correlation coefficient = 0.89) across the subgroups, confirming the reproducibility and structure of signal lags in white matter. We also applied a clustering analysis to identify functional networks within white matter. Analysis of latencies within and between networks revealed a similar pattern of inter- and intra-network communication to that reported for gray matter. Moreover, a unidirectional path, from inferior to superior regions, of BOLD signal propagation was revealed by higher resolution clustering. The variations of lag structure within white matter are associated with different sensory states (eyes open vs eyes closed, and eyes open with fixation vs. eyes closed). These findings provide additional insight into the character and roles of white matter BOLD signals in brain functions.Significance StatementFunctional MRI (fMRI) has had major impacts on clinical and basic neuroscience, and it has been used extensively to study the functional role and spatiotemporal organization of gray matter in different states. However, functional MRI signals from white matter have usually been ignored or even identified as artifacts. We used fMRI data from 1393 subjects to demonstrate (1) fMRI BOLD signals in white matter are robustly detectable in a resting state and exhibit a reproducible, spatiotemporal organization, similar to gray matter; (2) functional networks within white matter can be obtained by applying clustering analysis on the white matter connectivity matrix; (3) the pattern of signal latencies within and between networks resembles the results for gray matter. Further studies on the Beijing EOEC dataset II also revealed that the variations of latencies within white matter alter with different sensory (visual) states. Our findings demonstrate the that resting-state BOLD signals within white matter should be incorporated into comprehensive models of brain function.

Spatiotemporal organization of BamA governs the pattern of outer membrane protein distribution in growing Escherichia coli cells

The outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on β-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the β-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/μm2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth.SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the β-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cell’s growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.