In vertebrates, mutations in Protein O-mannosyltransferase1 (POMT1) or POMT2 are associated with muscular dystrophy due to a requirement for O-linked mannose glycans on the Dystroglycan (Dg) protein. In this study we examine larval body wall muscles of Drosophila mutant for Dg, or RNA interference knockdown for Dg and find defects in muscle attachment, altered muscle contraction, and a change in muscle membrane resistance. To determine if POMTs are required for Dg function in Drosophila, we examine larvae mutant for genes encoding POMT1 or POMT2. Larvae mutant for either POMT, or doubly mutant for both, show muscle attachment and muscle contraction phenotypes identical to those associated with reduced Dg function, consistent with a requirement for O-linked mannose on Drosophila Dg. Together these data establish a central role for Dg in maintaining integrity in Drosophila larval muscles and demonstrate the importance of glycosylation to Dg function in Drosophila. This study opens the possibility of using Drosophila to investigate muscular dystrophy.

BackgroundAlthough the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear.ResultsIn the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II.ConclusionOur results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

An earlier study has shown that RNAi knock-down of a single organic anion transporter (OAT) gene in the principal cells of Drosophila Malpighian tubules is associated with reductions in the expression of multiple, functionally related genes. In this study, we measured the rates of secretion of four fluorescent ions by tubules isolated from flies expressing targeted RNAi knock-down of specific OAT genes. Droplets secreted by isolated tubules set up in the Ramsay assay were collected in optically flat capillary tubes and the concentrations of fluorescent ions were determined by confocal laser scanning microscopy. Reductions in the expression of organic anion (OA) transporting polypeptide 58Dc (OATP; CG3380) were associated with reduced secretion of the OAs fluorescein and Texas Red. Reduction in the expression of Drosophila multidrug resistance associated protein (dMRP; CG6214) was correlated with reduced secretion of the P-glycoprotein substrate daunorubicin. Secretion of the organic cation quinacrine was unaffected by reduced expression of OATP, dMRP, or a multidrug efflux transporter (MET; CG30344). The results highlight the difficulties of assigning a rate-limiting role in transport of a specific OA to a single membrane transporter.

Seabrooke S, Stewart BA. Synaptic transmission and plasticity are modulated by nonmuscle myosin II at the neuromuscular junction of Drosophila. J Neurophysiol 105: 1966 -1976, 2011. First published February 16, 2011 doi:10.1152/jn.00718.2010.-The synaptic vesicle population in a nerve terminal is traditionally divided into subpopulations according to physiological criteria; the readily releasable pool (RRP), the recycling pool, and the reserve pool. It is recognized that the RRP subserves synaptic transmission evoked by low-frequency neural activity and that the recycling and reserve populations are called on to supply vesicles as neural activity increases. Here we investigated the contribution of nonmuscle myosin II (NMMII) to synaptic transmission with emphasis on the role a motor protein could play in the supply of vesicles. We used Drosophila genetics to manipulate NMMII and assessed synaptic transmission at the larval neuromuscular junction. We observed a positive correlation between synaptic strength at low-frequency stimulation and NMMII expression: reducing NMMII reduced the evoked response, while increasing NMMII increased the evoked response. Further, we found that NMMII contributed to the spontaneous release of vesicles differentially from evoked release, suggesting differential contribution to these two release mechanisms. By measuring synaptic responses under conditions of differing external calcium concentration in saline, we found that NMMII is important for normal synaptic transmission under high-frequency stimulation. This research identifies diverse functions for NMMII in synaptic transmission and suggests that this motor protein is an active contributor to the physiology of synaptic vesicle recruitment.electrophysiology; low-frequency depression; high frequency; vesicle pools SYNAPTIC VESICLES ARE DESCRIBED as existing in three populations depending on how they participate is synaptic transmission (Rizzoli and Betz 2005). The readily releasable pool (RRP) of vesicles contains those vesicles that are released immediately upon action potential arrival at the nerve terminal. The RRP typically makes up 1-2% of the total vesicle pool and is rapidly depleted by repetitive stimulation. The recycling pool, com-

The blood-brain barrier (BBB) physiologically isolates the brain from the blood and, thus, plays a vital role in brain homeostasis. Ion transporters play a critical role in this process by effectively regulating access of chemicals to the brain. Organic anion-transporting polypeptides (Oatps) transport a wide range of amphipathic substrates and are involved in efflux of chemicals across the vertebrate BBB. The anatomic complexity of the vascularized vertebrate BBB, however, creates challenges for experimental analysis of these processes. The less complex structure of the Drosophila BBB facilitates measurement of solute transport. Here we investigate a physiological function for Oatp58Dc in transporting small organic anions across the BBB. We used genetic manipulation, immunocytochemistry, and molecular techniques to supplement a whole animal approach to study the BBB. For this whole animal approach, the traceable small organic anion fluorescein was injected into the hemolymph. This research shows that Oatp58Dc is involved in maintaining a chemical barrier against fluorescein permeation into the brain. Oatp58Dc expression was found in the perineurial and subperineurial glia, as well as in postmitotic neurons. We specifically targeted knockdown of Oatp58Dc expression in the perineurial and subperineurial glia to reveal that Oatp58Dc expression in the perineurial glia is necessary to maintain the barrier against fluorescein influx into the brain. Our results show that Oatp58Dc contributes to maintenance of a functional barrier against fluorescein influx past the BBB into the brain.