Differential labelling of bulk endocytosis in nerve terminals by FM dyes

Bulk endocytosis is triggered in central nerve terminals during intense physiological stimulation. This endocytosis pathway can be labelled by the dye FM1-43 but not its more hydrophilic counterpart FM2-10. This selective labelling was proposed to be due to the retention of FM1-43, but not FM2-10, in slowly retrieving structures after washout of the dye. However, this explanation assumed that bulk endocytosis was a slow process that persisted after stimulation. We have recently shown that the great majority of bulk endocytosis occurs during stimulation, therefore another explanation for the specific labelling of this pathway by FM1-43 must be found. In this paper we show that the ability of FM dyes to label bulk endocytosis is dependent on the concentration of dye used and not their washout properties. When the loading concentration of FM1-43 was reduced 10-fold, its ability to label bulk endocytosis was lost. Conversely when the loading concentration of FM2-10 was increased 10-fold, it now labelled the pathway. This suggests that a difference in affinity of bulk endosome membranes for FM1-43 and FM2-10 underlies the disparity in labelling.     

Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand,but are defective in ligand uptake and degradation

The examination of insulin exocytosis at the single cell level by conventional electrophysiologic and amperometric methods possesses inherent limitations, and may not accurately reflect the morphologic events of exocytosis of the insulin granule. To overcome some of these limitations, we show by epifluorescent microscopy of a fluorescent dye, FM1-43, its incorporation into the plasma membrane and oncoming insulin granules undergoing exocytosis, and their core proteins. Using this method, we tracked exocytosis in real-time in insulinoma INS-1 and single rat islet beta cells in response to KCl and glucose. We observed both single transient and multi-stepwise increases in membrane FM1-43 fluorescence, suggesting single granule exocytosis as well as sequential and compound exocytosis, respectively. Confocal microscopy of nonpermeabilized cells shows that some of the exocytosed insulin granules labeled by the FM1-43 dye could also be labeled with insulin antibodies, suggesting prolonged openings of the fusion pores and slow dissolution of the granule core proteins on the membrane surface.     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Methods for detecting internalized, FM 1-43 stained particles in epithelial cells and monolayers

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.     

Cytochemical demonstration of acid phosphatase, trimetaphosphatase and basic protein in rat peritoneal mast cells during 48/80 induced exocytosis

Acid phosphatase (AcP-A), trimetaphosphatase (TmP-A) activities and basic protein reaction were cytochemically studied in rat peritoneal mast cells 15 minutes after stimulation by compound 48/80. The AcPase reaction was positive in slightly altered granules, but negative in those more intensely altered, and also in unaltered granules. The TmP-A reaction was negative in altered granules and positive in a few unaltered granules. These results suggest that mast cells have two populations of granules, one, comprising most granules, is AcP-A positive and is exocytosed. The other, smaller, is TmP-A positive, and is not exocytosed. Intact granules gave a strong positive reaction with amoniacal silver nitrate (AS), which detects basic protein. This reaction decreased in intensity with increasing granule alteration.     

Investigation vesicle cycle in nerve formations in somatic muscle of the earthworm Lumbricus terrestris

Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.     

Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.     

Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Here we show that beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired.     

Spatial variability in release at the frog neuromuscular junction measured with FM1-43

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye ¹; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K⁺). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A_i-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A_iv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining). Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy ^2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.Open in a separate windowClick here to view.^{(49M, flv)}     

The plasma membrane Q-SNARE syntaxin 2 enters the zymogen granule membrane during exocytosis in the pancreatic acinar cell

During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-mum diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.     

Changes in melanin granules in the fox due to coat color mutations

Hair samples from 11 different coat color phenotypes of the fox (Vulpes vulpes) were examined microscopically to determine the effects of several mutations on melanin granule color and distribution. Standard silver (b/b) causes the production of eumelanin rather than the phaeomelanin produced in wild-type red fox. Fromm brown (bf/bf) and Collicott brown (cb/cb) change the shape of the granules and convert eumelanin to brown and dark brown, respectively. The color dilution of Eastern Pearl (pe/pe) and Mansfield Pearl (pm/pm) is caused by clumping of granules in specific manners. Hairs from animals expressing more than one mutant gene, such as Amber (b/b pe/pe bf/bf), show the color and distribution of granules expected from interactions of independent loci.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules

The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.     

Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.     

Measuring exocytosis in neurons using FM labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release. FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated. Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence. Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.     

Quantitative evaluation of the uptake capacity of intact thrombocytes using fluorescent dyes

A new approach to quantitative determination of fluorescent dye uptake by intact cells is suggested. Fluorescent amine acridine orange selectively accumulating in 5HT granules of platelets has been used. Fluorescence signal analysis allows the estimation of a relative granule volume and the ratios of acridine orange transfer over cytoplasmic and granule membranes. The following results were obtained in human and rabbit platelets: a relative granule size was 14 +/- 1 % and 29 +/- 2 % of the total cell volume, intra-granule to extra-granule dye concentration ratios were 2260 +/- 382 and 30000 +/- 5550, while intra-cytoplasm to extra-cytoplasm concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.