Elsevier

Methods

Volume 33, Issue 4, August 2004, Pages 287-294
Methods

Using FM1-43 to study neuropeptide granule dynamics and exocytosis

https://doi.org/10.1016/j.ymeth.2004.01.002Get rights and content

Abstract

In the study of neuropeptide secretion and membrane trafficking, the fluorescent dye FM1-43 provides the ability to label selectively those structures that are undergoing exocytosis and endocytosis in living cells in real time. This review describes the unique properties of the FM dyes that make them ideal for studying neuropeptide granule dynamics and discusses various techniques that take advantage of FM dyes.

Introduction

Activity-dependent fluorescent dyes [1], [2] selectively stain secretory membrane structures that are undergoing exocytosis and endocytosis and have been used extensively to investigate synaptic vesicle recycling. In this review, we will focus on the use of FM1-43 and related dyes to study exocytosis not of synaptic vesicles, but of dense core granules, especially by prolactin-secreting pituitary lactotrophs. Whereas FM dyes typically stain only membranes, in pituitary lactotrophs, but not other neuroendocrine cells, the entire dense core secretory granule, which contains no lipid, stains with FM1-43 [3]. This apparently unique property of lactotroph dense core granules has enabled the monitoring of single exocytic events in living cells in real time with ordinary epifluorescence microscopy [3], [4]. We will describe the results of these studies as well as previously unpublished observations using FM dyes in combination with electron microscopy to investigate the post-exocytic fate of prolactin granules. We also consider other applications and caveats for the use of FM dyes.

Section snippets

FM1-43 and its variants

The family of fluorescent styryl compounds known as FM dyes has been useful for the study of exocytosis, endocytosis, and vesicle trafficking due to three properties: (1) FM dyes reversibly partition into membranes. When dye is applied to a preparation, all surface membranes become stained. When the preparation is washed with dye-free medium, the cell surface “destains” as dye molecules leave the plasma membrane. (2) FM dyes do not ordinarily “flip-flop” across membranes and therefore

Monitoring exocytosis

FM dyes have been used to study membrane dynamics in several peptide-secreting cells. Adrenal chromaffin cells [6] and pituitary somatotrophs [8], when stimulated in the presence of FM dye, exhibit diffuse plasma membrane brightening (Fig. 2). This increase in surface membrane fluorescence is proportional to the amount of vesicular membrane added during exocytosis. Thus, although resolution of individual granule fusion events in many endocrine cells is not achieved, monitoring FM fluorescence

GFP-tagged proteins

Green Fluorescent Protein (GFP)-tagged proteins have been used extensively to monitor secretory vesicle biogenesis, trafficking, and secretion [11]. If cells are transfected with a releasable GFP-tagged secretory granule protein and also stained with FM4-64, it is possible to monitor individual secretory granules before, during, and after membrane fusion. As a granule undergoes exocytosis, the GFP-tagged protein is released into the extracellular space and the green fluorescence of the granule

AM1-43 fixation combined with immunocytochemistry

To assess the colocalization of various proteins with stained granules, it is now possible to perform immunocytochemistry on fixed and permeabilized cells that have been stained with a modified form of FM1-43. AM1-43 (Biotium, Richmond, CA) is the modified form of FM1-43 in which an aldehyde-reactive amine has been added to the hydrophilic tail [13]. This offers the advantage of making AM1-43 more amenable than FM1-43 to standard aldehyde fixation and detergent permeabilization protocols used

FM1-43 photoconversion

While FM1-43 has been most extensively used to monitor membrane traffic in living preparations at the resolution of fluorescence microscopy, the ability to photoconvert the dye in the presence of diaminobenzidine (DAB) has enabled studies of FM1-43-stained membranes to be extended to the ultrastructural level. This technique has proven useful for identifying intermediates of the synaptic vesicle cycle [14], [15], [16], [17].

We photoconverted FM1-43 (Molecular Probes, Eugene, OR) stained dense

Other considerations

Although technical problems such as photobleaching and phototoxicity are encountered during live cell imaging with most fluorescent probes, some additional considerations should be taken into account when using FM dyes.

Summary

By using FM dyes to label the dense core granules of pituitary lactotrophs, we have been able to monitor optically single exocytic events in real time. This technique has allowed us to study the spatial organization of membrane fusion sites, examine signaling pathways involved in the regulation of compound exocytosis, and investigate the time course of exocytosis and endocytosis in these cells.

The utility of being able to stain dense cores with FM dyes has been enhanced by several techniques.

Acknowledgments

We thank Steven Fadul for excellent technical support. This work has been supported by NIH Grant DK 57999 to J.K.A and NIH Grants NS36665 and NS23466 to W.J.B. A.C.B is supported by an NIH Training Grant (T32 NS07083).

References (24)

  • J.K. Angleson et al.

    Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events

    Nat. Neurosci.

    (1999)
  • A.J. Cochilla et al.

    Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs

    J. Cell Biol.

    (2000)
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