Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.

The optical microscope has revolutionized biology since at least the 17 Century. Since then, it has progressed from a largely observational tool to a powerful bioanalytical platform. However, realizing its full potential to study live specimens is hindered by a daunting array of technical challenges. Here, we delve into the current state of live imaging to explore the barriers that must be overcome and the possibilities that lie ahead. We venture to envision a future where we can visualize and study everything, everywhere, all at once - from the intricate inner workings of a single cell to the dynamic interplay across entire organisms, and a world where scientists could access the necessary microscopy technologies anywhere.

SUMMARY: ImgLib2 is an open-source Java library for n-dimensional data representation and manipulation with focus on image processing. It aims at minimizing code duplication by cleanly separating pixel-algebra, data access and data representation in memory. Algorithms can be implemented for classes of pixel types and generic access patterns by which they become independent of the specific dimensionality, pixel type and data representation. ImgLib2 illustrates that an elegant high-level programming interface can be achieved without sacrificing performance. It provides efficient implementations of common data types, storage layouts and algorithms. It is the data model underlying ImageJ2, the KNIME Image Processing toolbox and an increasing number of Fiji-Plugins.

AVAILABILITY: ImgLib2 is licensed under BSD. Documentation and source code are available at http://imglib2.net and in a public repository at https://github.com/imagej/imglib.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics Online.

CONTACT: saalfeld@mpi-cbg.de

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a "decommissioning" phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the Mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages.

In vivo intracellular recordings of hippocampal neurons reveal the occurrence of fast events of small amplitude called spikelets or fast prepotentials. Because intracellular recordings have been restricted to anesthetized or head-fixed animals, it is not known how spikelet activity contributes to hippocampal spatial representations. We addressed this question in CA1 pyramidal cells by using in vivo whole-cell recording in freely moving rats. We observed a high incidence of spikelets that occurred either in isolation or in bursts and could drive spiking as fast prepotentials of action potentials. Spikelets strongly contributed to spiking activity, driving approximately 30% of all action potentials. CA1 pyramidal cell firing and spikelet activity were comodulated as a function of the animal’s location in the environment. We conclude that spikelets have a major impact on hippocampal activity during spatial exploration.

Neurons of the central nervous system (CNS) exhibit a variety of forms of synaptic plasticity, including associative long-term potentiation and depression (LTP/D), homeostatic activity-dependent scaling and distance-dependent scaling. Regulation of synaptic neurotransmitter receptors is currently thought to be a common mechanism amongst many of these forms of plasticity. In fact, glutamate receptor 1 (GluR1 or GluRA) subunits containing L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors have been shown to be required for several forms of hippocampal LTP and a particular hippocampal-dependent learning task. Because of this importance in associative plasticity, we sought to examine the role of these receptors in other forms of synaptic plasticity in the hippocampus. To do so, we recorded from the apical dendrites of hippocampal CA1 pyramidal neurons in mice lacking the GluR1 subunit (GluR1 -/-). Here we report data from outside-out patches that indicate GluR1-containing receptors are essential to the extrasynaptic population of AMPA receptors, as this pool was nearly empty in the GluR1 -/- mice. Additionally, these receptors appear to be a significant component of the synaptic glutamate receptor pool because the amplitude of spontaneous synaptic currents recorded at the site of input and synaptic AMPA receptor currents evoked by focal glutamate uncaging were both substantially reduced in these mice. Interestingly, the impact on synaptic weight was greatest at distant synapses such that the normal distance-dependent synaptic scaling used by these cells to counter dendritic attenuation was lacking in GluR1 -/- mice. Together the data suggest that the highly regulated movement of GluR1-containing AMPA receptors between extrasynaptic and synaptic receptor pools is critically involved in establishing two functionally diverse forms of synaptic plasticity: LTP and distance-dependent scaling.