Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals
ABSTRACT The styryl dye FM1-43 is a powerful tool to track exocytosis, endocytosis and recycling of secretory granules or vesicles. Due to its unique structure, dye molecules reversibly partition into the outer leaflet of surface membrane without permeating due to two cationic charges located in their headgroup. When a secretory cell is stimulated to evoke exocytosis, FM1-43 molecules that were inserted in the membrane are internalized during compensatory endocytosis and newly formed secretory granules or vesicles become stained with dye (staining/endocytosis). If stained secretory granules or vesicles undergo exocytosis in dye-free medium, due to concentration gradient, FM1-43 molecules dissociate from the membrane and loose fluorescence (destaining/exocytosis). Using a fluorescence microscope attached to a CCD camera or a confocal, it is possible to follow secretion in live cell or tissue preparations and in this chapter, we will make a description of FM1-43 staining and destaining protocol using the neuromuscular junction as experimental model. This technique has allowed answering important questions concerning synaptic vesicle recycling, which is a key step for neuronal communication. In addition, FM1-43 has proven to be an excellent tool for investigating membrane internalization and endosome recycling in a variety of cell types.
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ABSTRACT: Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury.International Journal of Nanomedicine 04/2012; 7:1977-89. DOI:10.2147/IJN.S30706 · 4.38 Impact Factor
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ABSTRACT: Homeostasis of adherens junctions is achieved through complex regulatory mechanisms. The junctions are highly dynamic in contact establishment, in remodeling events during development, and during processes involving a loss of adhesion like epithelial-mesenchyme transition. It appeared recently that they are also dynamically renewed in mature, steady-state adhesions. Indeed, maintenance of a steady state must be integrated into a tight control of force equilibrium between a cell and its neighbors. Therefore, it appears that E-cadherin dynamics allows to respond constantly to various biochemical and mechanical stimuli and to regulate the movement and shape of junctions in active remodeling processes. E-cadherin dynamics is mediated through several mechanisms (diffusion, trafficking) in function of the biological system. In mature junctions, membrane E-cadherin is quickly renewed by endocytosis in many cell types. E-cadherin endocytosis shows a complex regulation, depending on small G proteins, ubiquitination, cleavage events, actomyosin cytoskeleton, and other trans molecules in adherens junctions. It is modulated by growth factor stimulations and physical factors. Consequently, E-cadherin endocytosis tightly controls a number of functional processes: cell movements, junction maintenance, cell sorting, and polarity. Misregulated E-cadherin endocytosis is involved in many diseases, from cancerous processes to organogenesis defects.International review of cell and molecular biology 01/2012; 295:63-108. DOI:10.1016/B978-0-12-394306-4.00008-3 · 3.42 Impact Factor
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ABSTRACT: Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 μM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH(0.5)) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.AJP Cell Physiology 05/2012; 303(4):C376-84. DOI:10.1152/ajpcell.00067.2012 · 3.78 Impact Factor