We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not latestage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until ®ssion.

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.

Our data suggest a mechanism linking synaptic transmission and postsynaptic TGF-beta signaling that may coordinate nerve-muscle development and function.

Medulloblastoma (MB) is the most common brain cancer diagnosed among children. The cellular pathways that regulate MB invasion in response to environmental cues remain incompletely understood. Herein, we examine the migratory response of human MB-derived Daoy cells to different concentration profiles of Epidermal Growth Factor (EGF) using a microfluidic system. Our findings provide the first quantitative evidence that EGF concentration gradients modulate the chemotaxis of MB-derived cells in a dose-dependent manner via the EGF receptor (EGF-R). Data illustrates that higher concentration gradients caused increased number of cells to migrate. In addition, our results show that EGF-induced receptor phosphorylation triggered the downstream activation of phosphoinositide-3 kinase (PI3K)/Akt pathway, while its downstream activation was inhibited by Tarceva (an EGF-R inhibitor), and Wortmannin (a PI3K inhibitor). The treatment with inhibitors also severely reduced the number of MB-derived cells that migrated towards increasing EGF concentration gradients. Our results provide evidence to bolster the development of anti-migratory therapies as viable strategies to impede EGF-stimulated MB dispersal.

An increasing number of studies have demonstrated the multiple advantages of using nanocrystals, such as Quantum dots, for biological imaging. Quantum dots functionalized with biomolecules on their surfaces were shown to be able to bind to specific extracellular targets via specific recognition and to be internalized inside the cells, thereby allowing the imaging of intracellular pathways. However, the use of Quantum dots for live tracking of intracellular molecules is relatively limited because of the difficulties encountered during the induction of Quantum dots across living cell membranes. In this study we show that cationic liposomes can deliver low concentrations of non-targeted Quantum dots into the cytosol of living cells via a lipid-mediated fusion with the cell membrane. The intracellular Quantum dots exhibit aggregation that appears dependent upon their concentration, but does not visibly affect cell viability. Our results point towards the use of cationic liposomes as an effective delivery system for targeted Quantum dots within the cell cytosol, which would facilitate live cell imaging of the labeled molecules.