Annexin 2 has an essential role in actin-based macropinocytic rocketing.

Annexin 2 is a Ca(2+) binding protein that binds to and aggregates secretory vesicles at physiological Ca(2+) levels [1] and that also associates Ca(2+) independently with early endosomes [2, 3]. These properties suggest roles in both exocytosis and endocytosis, but little is known of the dynamics of Annexin 2 distribution in live cells during these processes. We have used evanescent field microscopy to image Annexin 2-GFP in live, secreting rat basophilic leukemia cells and in cells performing pinocytosis. Although we found no evidence of Annexin 2 involvement in exocytosis, we observed an enrichment of Annexin 2-GFP in actin tails propeling macropinosomes. The association of Annexin 2-GFP with rocketing macropinosomes was specific because Annexin 2-GFP was absent from the actin tails of rocketing Listeria. This finding suggests that the association of Annexin 2 with macropinocytic rockets requires native pinosomal membrane. Annexin 2 is necessary for the formation of macropinocytic rockets since overexpression of a dominant-negative Annexin 2 construct abolished the formation of these structures. The same construct did not prevent the movement of Listeria in infected cells. These results show that recruitment of Annexin 2 to nascent macropinosome membranes 16656is an essential prerequisite for actin polymerization-dependent vesicle locomotion.

A real-time view of life within 100 nm of the plasma membrane.

The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.

Transport, capture and exocytosis of single synaptic vesicles at active zones.

To sustain high rates of transmitter release, synaptic terminals must rapidly re-supply vesicles to release sites and prime them for exocytosis. Here we describe imaging of single synaptic vesicles near the plasma membrane of live ribbon synaptic terminals. Vesicles were captured at small, discrete active zones near the terminal surface. An electric stimulus caused them to undergo rapid exocytosis, seen as the release of a fluorescent lipid from the vesicles into the plasma membrane. Next, vesicles held in reserve about 20 nm from the plasma membrane advanced to exocytic sites, and became release-ready 250 ms later. Apparently a specific structure holds vesicles at an active zone to bring v-SNAREs and t-SNAREs, the proteins that mediate vesicle fusion, within striking distance of each other, and then allows the triggered movement of such vesicles to the plasma membrane.

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy.

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells.

Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.

Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy.

We have observed secretory granules beneath the plasma membrane of chromaffin cells. Using evanescent-field excitation by epiillumination, we have illuminated a thin layer of cytosol where cells adhere to glass coverslips. Up to 600 frames could be recorded at diffraction-limited resolution without appreciable photodynamic damage. We localized single granules with an uncertainty of approximately 30 nm and tracked their motion in three dimensions. Granules in resting cells wander randomly as if imprisoned in a cage that leaves approximately 70 nm space around a granule. The "cage" itself moves only slowly (D = 2 x 10(-12) cm2/s). Rarely do granules arrive at or depart from the plasma membrane of resting cells. Stimulation increases lateral motion only slightly. After the plasma membrane has been depleted of granules by exocytosis, fresh granules can be seen to approach it at an angle. The method will be useful for exploring the molecular steps preceding exocytosis at the level of single granules.

Ethane-freezing/methanol-fixation of cell monolayers: a procedure for improved preservation of structure and antigenicity for light and electron microscopies.

In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events, and organelle movements, it is necessary to fix/stop time-dependent events in the millisecond range. Ideally, immunoelectron microscopical labeling experiments require the availability of high-affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF) and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton.

Targeting of green fluorescent protein to neuroendocrine secretory granules: a new tool for real time studies of regulated protein secretion.

Human chromogranin B (hCgB), a soluble marker protein of neuroendocrine secretory granules, was fused to green fluorescent protein (GFP). Two GFP-mutants with different folding properties, S65T and EGFP, were used to produce two recombinant proteins, hCgB-GFP(S65T) and hCgB-EGFP, respectively. After transient expression only hCgB-EGFP elicited green fluorescence in the neuroendocrine cell line PC12. Pulse-chase experiments with [35S]sulfate followed by subcellular fractionation showed that hCgB-EGFP was sorted with high efficiency to immature secretory granules (ISG). Confocal microscopy revealed that fluorescent hCgB-EGFP colocalized largely with synaptotagmin, a membrane marker of secretory granules and synaptic-like microvesicles, and significantly with endogenous rat chromogranin B (rCgB), a soluble marker of secretory granules. Upon stimulation of transfected cells with 5 mM Ba2+ or by depolarization with 50 mM K+ hCgB-EGFP underwent regulated exocytosis. The dynamics of green fluorescent secretory granules beneath the plasma membrane (PM) of living PC12 cells were visualized by confocal microscopy. The majority of these vesicles did not move within 8.5 sec as if they were docked. In contrast, in NGF-induced cells most of the secretory granules beneath the somatic PM moved within the same time period whereas only little movement was observed in the neurites. These findings indicate that in differentiated PC12 cells the majority of the docking zones are not in the soma but are distributed along the neurites. In conclusion, the fusion protein hCgB-EGFP provides a powerful tool to study in real time vesicular traffic in the regulated pathway of protein secretion.

The exocytotic event in chromaffin cells revealed by patch amperometry.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.

Transport, docking and exocytosis of single secretory granules in live chromaffin cells.

Neurons maintain a limited pool of synaptic vesicles which are docked at active zones and are awaiting exocytosis. By contrast, endocrine cells releasing large, dense-core secretory granules have no active zones, and there is disagreement about the size and even the existence of the docked pool. It is not known how, and how rapidly, secretory vesicles are replaced at exocytic sites in either neurons or endocrine cells. By using electron microscopy, we have now been able to identify a pool of docked granules in chromaffin cells that is selectively depleted when cells secrete. With evanescent-wave fluorescence microscopy, we observed single granules undergoing exocytosis and leaving behind patches of bare plasmalemma. Fresh granules travelled to the plasmalemma at a top speed of 114 nm s(-1), taking an average of 6 min to arrive. On arrival, their motility diminished 4-fold, probably as a result of docking. Some granules detached and returned to the cytosol. We conclude that a large pool of docked granules turns over slowly, that granules move actively to their docking sites, that docking is reversible, and that the 'rapidly releasable pool' measured electrophysiologically represents a small subset of docked granules.

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein.

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy.

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.

Local Ca2+ release from internal stores controls exocytosis in pituitary gonadotrophs.

Exocytosis and the cell-averaged cytosolic [Ca2+], [Ca2+]i, were tracked in single gonadotrophs. Cells released 100 granules/s at 1 microM = [Ca2+]i when gonadotropin-releasing hormone (GnRH) activated IP3-mediated Ca2+ release from internal stores, but only 1 granule/s when [Ca2+]i was raised uniformly to 1 microM by other means. Strong exocytosis was then seen only at higher [Ca2+]i (half-maximal at 16 microM). Parallel second messengers did not contribute to GnRH-induced exocytosis, because IP3 alone was as effective as GnRH, and because even GnRH failed to trigger rapid exocytosis when the [Ca2+]i rise was blunted by EGTA. When [Ca2+]i was released from stores, exocytosis depended on [Ca2+]i rising rapidly, as if governed by Ca2+ flux into the cytosol. We suggest that IP3 releases Ca2+ selectively from subsurface cisternae, raising [Ca2+] near exocytic sites 5-fold above the cell average.

Ca2+ triggers massive exocytosis in Chinese hamster ovary cells.

We have tracked the cell surface area of CHO cells by measuring the membrane capacitance, Cm. An increase in cytosolic [Ca2+], [Ca2+]i, increased the cell surface area by 20-30%. At micromolar [Ca2+]i the increase occurred in minutes, while at 20 microM or higher [Ca2+]i it occurred in seconds and was transient. GTPgammaS caused a 3% increase even at 0.1 microM [Ca2+]i. We conclude that CHO cells, previously thought capable only of constitutive exocytosis, can perform Ca2+-triggered exocytosis that is both massive and rapid. Ca2+-triggered exocytosis was also observed in 3T3 fibroblasts. Our findings add evidence to the view that Ca induces exocytosis in cells other than known secretory cells.

Fast steps in exocytosis and endocytosis studied by capacitance measurements in endocrine cells.

The past year has witnessed progress in identifying late steps in exocytosis that are so short-lived as to be difficult to study biochemically. Recent studies have also revealed a novel and surprisingly fast mechanism of endocytosis that may be triggered by a rise in the intracellular concentration of Ca2+ and that retrieves exocytosed membrane in seconds.

Millisecond studies of calcium-dependent exocytosis in pituitary melanotrophs: comparison of the photolabile calcium chelators nitrophenyl-EGTA and DM-nitrophen.

DM-nitrophen (DMN) is a photolabile calcium chelator that has been used extensively to study calcium-triggered exocytosis. Nitrophenyl-EGTA (NPE) is a recently synthesized photolabile calcium chelator that, unlike DMN, selectively binds calcium over magnesium. Here, we compare NPE and DMN for their effectiveness in raising cytosolic calcium ([Ca]i) to trigger exocytosis. The whole cell patch clamp technique was used to monitor membrane capacitance (Cm) and to load both calcium indicator dye and photolabile chelators into rat pituitary melanotrophs prior to flash photolysis. In cells dialysed with DMN, a transient increase in [Ca]i was observed immediately after continuity between the patch pipette and the cell cytosol was achieved. This 'loading transient' reflects the release of calcium from DMN during the binding of intracellular magnesium. No such transient was seen with NPE, consistent with the negligible binding of magnesium to this chelator. Following flash photolysis of DMN or NPE, [Ca]i increased, triggering both a rapid exocytic burst and slower sustained phases of exocytosis. When flashes of the same intensity were compared, the photolysis of NPE resulted in smaller increases in [Ca]i and slower exocytic bursts than that of DMN. These findings are in accordance with the properties of the two compounds [Ellis-Davies G.C.R., Kaplan J.H. Nitrophenyl-EGTA, a photolabile chelator that selectively binds Ca2+ with high affinity and releases it rapidly upon photolysis. Proc Natl Acad Sci USA 1994; 91: 187-191] and the calcium dependency of the exocytic burst [Thomas P., Wong J.G., Lee A.K., Almers W. A low affinity Ca2+ receptor controls the final steps in peptide secretion from pituitary melanotrophs. Neuron 1993; 11: 93-104]. Although NPE is somewhat less effective than DMN in raising [Ca]i, this chelator promises to be a useful and interesting tool for the time-resolved study of calcium-dependent exocytosis in the presence of physiological concentrations of magnesium.

Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells.

Ca(2+)-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma in quickly frozen unfixed sections (3300 in a melanotroph and 830 in a chromaffin cell). Most exocytosis required tens of seconds, but a small pool of granules was released in tens of milliseconds. Evidently, only a small subset of docked granules is rapidly releasable. We suggest that, temporally, the last ATP-dependent step in exocytosis is closely associated with docking and that docked granules reach fusion competence only after subsequent steps.