Using the Fluorescent Styryl Dye FM1-43 to Visualize Synaptic Vesicles Exocytosis and Endocytosis in Motor Nerve Terminals

Department of Morphology, ICB, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 689:137-48. DOI: 10.1007/978-1-60761-950-5_8
Source: PubMed


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.

76 Reads
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine) or the whole ΔµH+ (bafilomycin), a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05). Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.
    Brazilian Journal of Pharmaceutical Science 03/2012; 48(1):155-161. DOI:10.1590/S1984-82502012000100017 · 0.26 Impact Factor
Show more