Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

Messenger RNA decay measurements are typically performed on a population of cells. However, this approach cannot reveal sufficient complexity to provide information on mechanisms that may regulate mRNA degradation, possibly on short timescales. To address this deficiency, we measured cell cycle-regulated decay in single yeast cells using single-molecule FISH. We found that two genes responsible for mitotic progression, SWI5 and CLB2, exhibit a mitosis-dependent mRNA stability switch. Their transcripts are stable until mitosis, when a precipitous decay eliminates the mRNA complement, preventing carryover into the next cycle. Remarkably, the specificity and timing of decay is entirely regulated by their promoter, independent of specific cis mRNA sequences. The mitotic exit network protein Dbf2p binds to SWI5 and CLB2 mRNAs cotranscriptionally and regulates their decay. This work reveals the promoter-dependent control of mRNA stability, a regulatory mechanism that could be employed by a variety of mRNAs and organisms.

AIM: Ethanol-induced locomotor sensitization is a behavioral manifestation of physiological responses to repeated ethanol exposures. While ethanol exerts direct effects on multiple neurotransmitter systems in the brain, ethanol-induced changes in metabolic state, including acute hyperglycemia and inhibition of insulin signaling, also have plausible roles in the expression of ethanol-related behaviors through direct and indirect effects on brain function. The current experiments examined whether insulin administration or the resultant hypoglycemia might attenuate the development of sensitization to the locomotor stimulant effect of ethanol.

MAIN METHODS: Male and female DBA/2J mice received daily injections of 5 or 10 IU/kg insulin before or after a stimulating dose of ethanol and subsequent testing in an automated activity monitor. Blood glucose levels were determined upon the completion of the experiments.

KEY FINDINGS: Insulin injected prior to ethanol blunted the acute stimulant response as well as the acquisition and expression of locomotor sensitization, while insulin given after ethanol did not affect the development of the sensitized response. In a separate experiment, mice given glucose concurrently with insulin developed ethanol-induced locomotor sensitization normally.

SIGNIFICANCE: These experiments suggest that insulin attenuates the development of ethanol-induced locomotor sensitization, and that blood glucose levels can largely account for this effect. Further studies of the role of ethanol-induced metabolic states should provide novel information on the expression of ethanol-related behaviors.

An agglomerative clustering algorithm merges the most similar pair of clusters at every iteration. The function that evaluates similarity is traditionally hand- designed, but there has been recent interest in supervised or semisupervised settings in which ground-truth clustered data is available for training. Here we show how to train a similarity function by regarding it as the action-value function of a reinforcement learning problem. We apply this general method to segment images by clustering superpixels, an application that we call Learning to Agglomerate Superpixel Hierarchies (LASH). When applied to a challenging dataset of brain images from serial electron microscopy, LASH dramatically improved segmentation accuracy when clustering supervoxels generated by state of the boundary detection algorithms. The naive strategy of directly training only supervoxel similarities and applying single linkage clustering produced less improvement.

Using the MS2 system for labeling mRNA, in this issue, Gallardo et al. (2011) find that telomere lengthening depends on a stable accumulation of multiple telomerase complexes in late S phase and that this process is temporally regulated by Rif1/2 proteins.

We provide evidence for a prodegenerative, glial-derived signaling framework in the Drosophila neuromuscular system that includes caspase and mitochondria-dependent signaling. We demonstrate that Drosophila TNF-α (eiger) is expressed in a subset of peripheral glia, and the TNF-α receptor (TNFR), Wengen, is expressed in motoneurons. NMJ degeneration caused by disruption of the spectrin/ankyrin skeleton is suppressed by an eiger mutation or by eiger knockdown within a subset of peripheral glia. Loss of wengen in motoneurons causes a similar suppression providing evidence for glial-derived prodegenerative TNF-α signaling. Neither JNK nor NFκβ is required for prodegenerative signaling. However, we provide evidence for the involvement of both an initiator and effector caspase, Dronc and Dcp-1, and mitochondrial-dependent signaling. Mutations that deplete the axon and nerve terminal of mitochondria suppress degeneration as do mutations in Drosophila Bcl-2 (debcl), a mitochondria-associated protein, and Apaf-1 (dark), which links mitochondrial signaling with caspase activity in other systems.

Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.

We describe a localization microscopy analysis method that is able to extract results in live cells using standard fluorescent proteins and xenon arc lamp illumination. Our Bayesian analysis of the blinking and bleaching (3B analysis) method models the entire dataset simultaneously as being generated by a number of fluorophores that may or may not be emitting light at any given time. The resulting technique allows many overlapping fluorophores in each frame and unifies the analysis of the localization from blinking and bleaching events. By modeling the entire dataset, we were able to use each reappearance of a fluorophore to improve the localization accuracy. The high performance of this technique allowed us to reveal the nanoscale dynamics of podosome formation and dissociation throughout an entire cell with a resolution of 50 nm on a 4-s timescale.

We tested whether Drosophila larvae can associate odours with a mechanosensory disturbance as a punishment, using substrate vibration conveyed by a loudspeaker (buzz:). One odour (A) was presented with the buzz, while another odour (B) was presented without the buzz (A/B training). Then, animals were offered the choice between A and B. After reciprocal training (A/B), a second experimental group was tested in the same way. We found that larvae show conditioned escape from the previously punished odour. We further report an increase of associative performance scores with the number of punishments, and an increase according to the number of training cycles. Within the range tested (between 50 and 200 Hz), however, the pitch of the buzz does not apparently impact associative success. Last, but not least, we characterized odour-buzz memories with regard to the conditions under which they are behaviourally expressed--or not. In accordance with what has previously been found for associative learning between odours and bad taste (such as high concentration salt or quinine), we report that conditioned escape after odour-buzz learning is disabled if escape is not warranted, i.e. if no punishment to escape from is present during testing. Together with the already established paradigms for the association of odour and bad taste, the present assay offers the prospect of analysing how a relatively simple brain orchestrates memory and behaviour with regard to different kinds of 'bad' events.

Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.