Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.

Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

GLD2 poly(A) polymerase is required for long-term memory.

The formation of long-term memory is believed to require translational control of localized mRNAs. In mammals, dendritic mRNAs are maintained in a repressed state and are activated upon repetitive stimulation. Several regulatory proteins required for translational control in early development are thought to be required for memory formation, suggesting similar molecular mechanisms. Here, using Drosophila, we identify the enzyme responsible for poly(A) elongation in the brain and demonstrate that its activity is required specifically for long-term memory. These findings provide strong evidence that cytoplasmic polyadenylation is critical for memory formation, and that GLD2 is the enzyme responsible.

cAMP-response element-binding protein and heat-shock protein 70 additively suppress polyglutamine-mediated toxicity in Drosophila.

Gene-specific expansion of polyglutamine-encoding CAG repeats can cause neurodegenerative disorders, including Huntington's disease. It is believed that part of the pathological effect of the expanded protein is due to transcriptional dysregulation. Using Drosophila as a model, we show that cAMP-response element-binding protein (CREB) is involved in expanded polyglutamine-induced toxicity. A mutation in the Drosophila homolog of CREB, dCREB2, enhances lethality due to polyglutamine peptides (polyQ), and an additional copy of dCREB2 partially rescues this lethality. Neuronal expression of expanded polyQ attenuates in vivo CRE-mediated transcription of a reporter gene. As reported previously, overexpression of heat-shock protein 70 (Hsp70) rescues polyglutamine-dependent lethality. However, it does not rescue CREB-mediated transcription. The protective effects of CREB and heat-shock protein 70 against polyQ are additive, suggesting that targeting multiple pathways may be effective for treatment of polyglutamine diseases.

New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants.

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. Here we show that at Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1Brelated protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, we show that Baz and Par-6 are also expressed at synapses and that their synaptic localization depends on aPKC activity. Our findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory.

Memory enhancement and formation by atypical PKM activity in Drosophila melanogaster.

Synaptic stimulation activates signal transduction pathways, producing persistently active protein kinases. PKMzeta is a truncated, persistently active isoform of atypical protein kinase C-zeta (aPKCzeta), which lacks the N-terminal pseudosubstrate regulatory domain. Using a Pavlovian olfactory learning task in Drosophila, we found that induction of the mouse aPKMzeta (MaPKMzeta) transgene enhanced memory. The enhancement required persistent kinase activity and was temporally specific, with optimal induction at 30 minutes after training. Induction also enhanced memory after massed training and corrected the memory defect of radish mutants, but did not improve memory produced by spaced training. The 'M' isoform of the Drosophila homolog of MaPKCzeta (DaPKM) was present and active in fly heads. Chelerythrine, an inhibitor of PKMzeta, and the induction of a dominant-negative MaPKMzeta transgene inhibited memory without affecting learning. Finally, induction of DaPKM after training also enhanced memory. These results show that atypical PKM is sufficient to enhance memory in Drosophila and suggest that it is necessary for normal memory maintenance.