It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

Many mammals forage and burrow in dark constrained spaces. Touch through facial whiskers is important during these activities, but the close quarters makes whisker deployment challenging. The diverse shapes of facial whiskers reflect distinct ecological niches. Rodent whiskers are conical, often with a remarkably linear taper. Here we use theoretical and experimental methods to analyze interactions of mouse whiskers with objects. When pushed into objects, conical whiskers suddenly slip at a critical angle. In contrast, cylindrical whiskers do not slip for biologically plausible movements. Conical whiskers sweep across objects and textures in characteristic sequences of brief sticks and slips, which provide information about the tactile world. In contrast, cylindrical whiskers stick and remain stuck, even when sweeping across fine textures. Thus the conical whisker structure is adaptive for sensor mobility in constrained environments and in feature extraction during active haptic exploration of objects and surfaces. DOI: http://dx.doi.org/10.7554/eLife.01350.001.

The ability to control the activity of specific neurons in freely behaving animals provides an effective way to probe the contributions of neural circuits to behavior. Wide interest in studying principles of neural circuit function using the fruit fly Drosophila melanogaster has fueled the construction of an extensive transgenic toolkit for performing such neural manipulations. Here we describe approaches for using these tools to manipulate the activity of specific neurons and assess how those manipulations impact the behavior of flies. We also describe methods for examining connectivity among multiple neurons that together form a neural circuit controlling a specific behavior. This work provides a resource for researchers interested in examining how neurons and neural circuits contribute to the rich repertoire of behaviors performed by flies.

Short-term memory is associated with persistent neural activity that is maintained by positive feedback between neurons. To explore the neural circuit motifs that produce memory-related persistent activity, we measured coupling between functionally characterized motor cortex neurons in mice performing a memory-guided response task. Targeted two-photon photostimulation of small (<10) groups of neurons produced sparse calcium responses in coupled neurons over approximately 100 μm. Neurons with similar task-related selectivity were preferentially coupled. Photostimulation of different groups of neurons modulated activity in different subpopulations of coupled neurons. Responses of stimulated and coupled neurons persisted for seconds, far outlasting the duration of the photostimuli. Photostimuli produced behavioral biases that were predictable based on the selectivity of the perturbed neuronal population, even though photostimulation preceded the behavioral response by seconds. Our results suggest that memory-related neural circuits contain intercalated, recurrently connected modules, which can independently maintain selective persistent activity.

Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.

Neuronal connectivity in the circadian clock network is essential for robust endogenous timekeeping. In the Drosophila circadian clock network, the four pairs of small ventral lateral neurons (sLNvs) serve as main pacemakers. Peptidergic communication via sLNv, which release the key output neuropeptide, Pigment Dispersing Factor (PDF), has been well characterized. In the absence of PDF, flies become largely arrhythmic, similar to the phenotype associated with the loss of the mammalian circadian peptide, VIP. In contrast, little is known about the role of the synaptic connections that sLNvs form with downstream neurons. Connectomic analyses revealed that despite their role as key pacemaker neurons within the clock network, the sLNvs form few connections with other clock neurons. However, they form strong synaptic connections with a small group of previously uncharacterized neurons, SLP316, which in turn synapse onto dorsal clock neurons. Here, we show that silencing SLP316 neurons via tetanus toxin (TNT) expression shortens the free-running period, whereas hyper-exciting them by expressing the constitutively open Na[+] channel, NaChBac, results in period lengthening. Under light-dark cycles, silencing SLP316 neurons also causes lower daytime activity and higher daytime sleep. Our results revealed that the main postsynaptic partners of the Drosophila pacemaker neurons are a non-clock neuronal cell type that regulates the timing of sleep and activity.

Taste detection and hunger state dynamically regulate the decision to initiate feeding. To study how context-appropriate feeding decisions are generated, we combined synaptic resolution circuit reconstruction with targeted genetic access to specific neurons to elucidate a gustatory sensorimotor circuit for feeding initiation in Drosophila melanogaster. This circuit connects gustatory sensory neurons to proboscis motor neurons through three intermediate layers. Most of the neurons in this pathway are necessary and sufficient for proboscis extension, a feeding initiation behavior, and respond selectively to sugar taste detection. Hunger signals act at select second-order neurons to increase feeding initiation in food-deprived animals. In contrast, a bitter taste pathway inhibits premotor neurons, illuminating a central mechanism that weighs sugar and bitter tastes to promote or inhibit feeding. Together, these studies reveal the neural circuit basis for the integration of external taste detection and internal nutritive state to flexibly execute a critical feeding decision.

In cap-dependent translation initiation, the open reading frame (ORF) of mRNA is established by the placement of the AUG start codon and initiator tRNA in the ribosomal peptidyl (P) site. Internal ribosome entry sites (IRESs) promote translation of mRNAs in a cap-independent manner. We report two structures of the ribosome-bound Taura syndrome virus (TSV) IRES belonging to the family of Dicistroviridae intergenic IRESs. Intersubunit rotational states differ in these structures, suggesting that ribosome dynamics play a role in IRES translocation. Pseudoknot I of the IRES occupies the ribosomal decoding center at the aminoacyl (A) site in a manner resembling that of the tRNA anticodon-mRNA codon. The structures reveal that the TSV IRES initiates translation by a previously unseen mechanism, which is conceptually distinct from initiator tRNA-dependent mechanisms. Specifically, the ORF of the IRES-driven mRNA is established by the placement of the preceding tRNA-mRNA-like structure in the A site, whereas the 40S P site remains unoccupied during this initial step.