BACKGROUND: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants.

METHODS: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure.

RESULTS: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects.

CONCLUSIONS: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

Commentary: The first example of true live cell and time lapse imaging by localization microscopy (as opposed to particle tracking), this paper uses the Nyquist criterion to establish a necessary condition for true spatial resolution based on the density of localized molecules – a condition often unmet in claims elsewhere in the superresolution literature.
By any method, higher spatiotemporal resolution requires increasing light exposure at the specimen, making noninvasive imaging increasingly difficult. Here, simultaneous differential interference contrast imaging is used to establish that cells behave physiologically before, during, and after PALM imaging. Similar controls are lacking from many supposed “live cell” superresolution demonstrations.

Microscopic resolution far beyond the diffraction limit is possible by localizing single molecules individually. This approach has now been demonstrated on living cells.

In a forward genetic screen for mutations that destabilize the neuromuscular junction, we identified a novel long isoform of Drosophila ankyrin2 (ank2-L). We demonstrate that loss of presynaptic Ank2-L not only causes synapse disassembly and retraction but also disrupts neuronal excitability and NMJ morphology. We provide genetic evidence that ank2-L is necessary to generate the membrane constrictions that normally separate individual synaptic boutons and is necessary to achieve the normal spacing of subsynaptic protein domains, including the normal organization of synaptic cell adhesion molecules. Mechanistically, synapse organization is correlated with a lattice-like organization of Ank2-L, visualized using extended high-resolution structured-illumination microscopy. The stabilizing functions of Ank2-L can be mapped to the extended C-terminal domain that we demonstrate can directly bind and organize synaptic microtubules. We propose that a presynaptic Ank2-L lattice links synaptic membrane proteins and spectrin to the underlying microtubule cytoskeleton to organize and stabilize the presynaptic terminal.

A recent study challenges the oft-held notion that ester bonds in prodrug molecules are cleaved rapidly and completely inside cells by endogenous, nonspecific esterases. Structure-activity relationship studies on acylated sugars reveal that regioisomeric compounds display disparate biological activity, suggesting that ester bonds can persist in a cellular context.

The Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF has the ability to alter chromatin structure and regulate transcription. Previous studies have shown that mutation of Drosophila NURF induces melanotic tumors, implicating NURF in innate immune function. Here, we show that NURF mutants exhibit identical innate immune responses to gain-of-function mutants in the Drosophila JAK/STAT pathway. Using microarrays, we identify a common set of target genes that are activated in both mutants. In silico analysis of promoter sequences of these defines a consensus regulatory element comprising a STAT-binding sequence overlapped by a binding-site for the transcriptional repressor Ken. NURF interacts physically and genetically with Ken. Chromatin immunoprecipitation (ChIP) localizes NURF to Ken-binding sites in hemocytes, suggesting that Ken recruits NURF to repress STAT responders. Loss of NURF leads to precocious activation of STAT target genes.

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

The development of high-resolution microscopy makes possible the high-throughput screening of cellular information, such as gene expression at single cell resolution. One of the critical enabling techniques yet to be developed is the automatic recognition or annotation of specific cells in a 3D image stack. In this paper, we present a novel graph-based algorithm, ARC, that determines cell identities in a 3D confocal image of C. elegans based on their highly stereotyped arrangement. This is an essential step in our work on gene expression analysis of C. elegans at the resolution of single cells. Our ARC method integrates both the absolute and relative spatial locations of cells in a C. elegans body. It uses a marker-guided, spatially-constrained, two-stage bipartite matching to find the optimal match between cells in a subject image and cells in 15 template images that have been manually annotated and vetted. We applied ARC to the recognition of cells in 3D confocal images of the first larval stage (L1) of C. elegans hermaphrodites, and achieved an average accuracy of 94.91%.

The ad hoc genetic correlation between ethanol sensitivity and learning mechanisms in Drosophila could overemphasize a common process supporting both behaviors. To challenge directly the hypothesis that these mechanisms are singular, we examined the learning phenotypes of 10 new strains. Five of these have increased ethanol sensitivity, and the other 5 do not. We tested place and olfactory memory in each of these lines and found two new learning mutations. In one case, altering the tribbles gene, flies have a significantly reduced place memory, elevated olfactory memory, and normal ethanol response. In the second case, mutation of a gene we name ethanol sensitive with low memory (elm), place memory was not altered, olfactory memory was sharply reduced, and sensitivity to ethanol was increased. In sum, however, we found no overall correlation between ethanol sensitivity and place memory in the 10 lines tested. Furthermore, there was a weak but nonsignificant correlation between ethanol sensitivity and olfactory learning. Thus, mutations that alter learning and sensitivity to ethanol can occur independently of each other and this implies that the set of genes important for both ethanol sensitivity and learning is likely a subset of the genes important for either process.

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.