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. 2005 Sep 8;568(Pt 3):917–929. doi: 10.1113/jphysiol.2005.094011

Exocytosis and endocytosis of small vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

Ting-Ting Liu 1,4, Takuya Kishimoto 1,2, Hiroyasu Hatakeyama 1, Tomomi Nemoto 1,3, Noriko Takahashi 1,3, Haruo Kasai 1,2
PMCID: PMC1464175  PMID: 16150796

Abstract

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) in the lateral membranes not facing the glass-cover slip. Upon photolysis of a caged Ca2+ compound, TEP imaging with FM1-43 (a polar membrane tracer) detected massive exocytosis of vesicles with a time constant of about 1 s. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that the diameter of vesicles was small (55 nm). Extensive exocytosis of small vesicles (SVs) was shown to be mediated by the transient opening of a fusion pore with a diameter less than about 1.6 nm, and to be followed by direct (‘kiss-and-run’) endocytosis and translocation of the endocytic vesicles (EVs) deep into the cytoplasm. These processes were unaffected by GTP-γ-S. In contrast, constitutive endocytic vesicles exhibited a diameter of 90 nm, took up molecules with a diameter of > 12 nm, and their formation was blocked by GTP-γ-S. Electron-microscopic investigation with photoconversion of diaminobenzidine using FM1-43 confirmed an abundance of EVs with a diameter of 54 nm in stimulated cells. They rapidly translocated into the cytosol, and fused with endosomal organelles. The number of SV exocytosis events vastly exceeded the number of SVs morphologically docked at the plasma membrane. Simultaneous capacitance and FM1-43 measurements indicated that TEP imaging detected most SV exocytosis, and the fusion pore was closed within 2 s. Thus, we have, for the first time, directly visualized massive exocytosis of small vesicles in a non-synaptic preparation, and have revealed their fusion-pore mediated exocytosis and endocytosis.

The formation of a fusion pore plays a central role in the exocytosis of secretory vesicles (Chandler & Heuser, 1980; Breckenridge & Almers, 1987; Alvarez de Toledo et al. 1993; Takahashi et al. 2002). Capacitance and amperometric measurements have revealed that the initial fusion pore is only 1.5 nm in diameter and that its opening is reversible (Neher & Marty, 1982; Breckenridge & Almers, 1987; Zimmerberg et al. 1987; Alvarez de Toledo et al. 1993; Albillos et al. 1997). Measurement of capacitance, however, does not allow the monitoring of a fusion pore with a conductance larger than the admittance of the vesicle membrane (Albillos et al. 1997; Klyachko & Jackson, 2002), and fusion pores larger than 1.0 nm in diameters cannot be investigated for small vesicles. Conversely, fusion pores with a diameter of < 4 nm are not detectable by electron microscopy (Chandler & Heuser, 1980; Kanaseki et al. 1997). Moreover, these techniques do not provide direct information on vesicle fates after closure of a fusion pore. Properties of fusion pores in small vesicles' exocytosis and subsequent endocytic pathways have thus been difficult to study (Valtorta et al. 2001; Jarousse & Kelly, 2001), and the existence of so-called ‘kiss-and-run’ endocytosis mediated by closure of fusion pores has remained controversial for small vesicles (Gandhi & Stevens, 2003; Aravanis et al. 2003b; Ryan, 2003; Rizzoli & Betz, 2004).

Exocytosis of small vesicles (SVs) is responsible for neurotransmitter release from presynaptic terminals. In addition, Ca2+-dependent exocytosis of SVs has been thought to be operative generally in eukaryotic cells and to contribute both to the recycling of membrane proteins (Cameron et al. 1991; Borgonovo et al. 2002) and to membrane repair (McNeil & Steinhardt, 2003). The existence of SV exocytosis in non-synaptic preparations has not been firmly established, however, because of the difficulties in characterizing this process in preparations without postsynaptic readout. For example, cells of the rat pheochromocytoma line PC12 possess SVs that contain acetylcholine (ACh) in addition to large dense-core vesicles (LVs) that contain monoamines (Schubert et al. 1977; Linstedt & Kelly, 1991; Cameron et al. 1991; Bauerfeind et al. 1993). Quantal secretion of ACh, however, accounts only for a small fraction of exocytosis in these cells (Ninomiya et al. 1997), and the actual contribution of SVs to Ca2+-dependent exocytosis has not been elucidated.

In this study we have applied TEP imaging and TEPIQ analysis to PC12 cells to investigate exocytosis and endocytosis of SVs in PC12 cells. We found an abundance of Ca2+-dependent exocytosis of SVs with a diameter of 55 nm. The fusion pore of SVs did not enlarge to more than 1.4 nm, and was closed within 2 s to generate direct endocytic vesicles. These endocytic vesicles rapidly detached from the plasma membrane, in line with the terminology of ‘kiss-and-run’ endocytosis. Electron microscopic investigations of photo-converted preparations identified an abundance of cytosolic small endocytic vesicles responsible for direct endocytosis of SVs, and, for the first time, revealed that they were rapidly transported to endosomal organelles. Membrane capacitance measurements confirmed that TEP imaging could detect most exocytosis and reported representative features of exo- and endocytosis of SVs in PC12 cells.

Methods

Cell preparation and TEP imaging

PC12 cells were grown in a Dulbecco's modified Eagle's medium(DME)-based culture medium in the absence of NGF (nerve growth factor) (Kishimoto et al. 2001), and used for physiological experiments as described (Kishimoto et al. 2005). The cells were subjected to local application with a glass pipette of one or two of the fluorescent tracers FM1-43 (25 μm), sulforhodamine-B (SRB, 0.5 mm), pyranine (5 mm), bis-fura2 (2 mm), Oregon Green 488–BAPTA-2 (2 mm), and fluorescein dextrans (2 mm), all of which were obtained from Molecular Probes. For washout of dyes, the recording chamber was rapidly superfused with solution lacking dye. The PC12 cells were loaded with 30 μm nitrophenyl-EGTA (NPE)-acetoxymethyl ester (AM) (Molecular Probes). Photolysis of NPE was induced with a mercury lamp (U-ULS100HG, Olympus) through a 360 nm band pass filter (Kasai et al. 1996).

The fluorescence of SRB was measured at 570–650 nm (red channel), whereas that of FM1-43, fura-2FF, 10 kDa fluorescein dextrans (FD), pyranine, bis-fura2, and Oregon Green 488–BAPTA-2 was measured at 400–550 nm (blue channel). For multicolour two-photon excitation imaging, overlap of the emission spectrum of one tracer with that of the other was estimated by measurement of the preparation stained with only one tracer under each experimental condition as described previously (Kasai et al. 2005).

TEPIQ analyses

TEPIQ analyses were conducted as described previously (Kasai et al. 2005; Kishimoto et al. 2005). The molecular diameters of pyranine (455 Da), SRB (558 Da), bis-fura2 (773 Da), and Oregon Green 488–BAPTA-2 (1287 Da) are considered to be 1.0, 1.4, 1.6 and 2.0 nm, respectively, given that the second-largest dimension is critical for pore permeation and that the molecular dimensions of these compounds are estimated to be 0.60 nm × 0.99 nm × 1.02 nm, 0.60 nm × 1.38 nm × 1.66 nm, 1.33 nm × 1.60 nm × 1.91 nm, and 1.62 nm × 2.03 nm × 2.47 nm, respectively (Chemi-3D Pro 6.0; Cambridge Soft, Cambridge, MA, USA). The hydrodynamic diameters of dextrans are assumed to be 4, 6 and 12 nm for 3, 10 and 70 kDa species, respectively (Ioan et al. 2000; Takahashi et al. 2002). These tracers contain negative charges: pyranine, three negative charges; SRB, one positive and one negative charge; bis-fura2, six negative charges; Oregon Green 488–BAPTA-2, eight negative charges; 3 kDa FD, 1.4 negative charges.

Simultaneous measurement of membrane capacitance and FM1-43 fluorescence

Single PC12 cells were observed with a UApo 40×, 340 oil objective lens (Olympus), and photolysis of NPE was induced with a xenon flash lamp (High Tech Instrument, Aberdeen, UK) (Kasai et al. 1996). We continuously applied FM1-43 from a local glass pipette, with the exception of the experiment shown in Fig. 8G, in which FM1-43 was added rapidly (< 4 ms) to the cell with a piezoelectric device (ASB003A; NEC, Tokyo, Japan). Whole-cell fluorescence of FM1-43 was elicited with a xenon lamp (ULS75XE, Olympus) at 475 nm, measured at 520–570 nm, corrected by subtraction of background fluorescence, and normalized by the background-corrected value for the cell before stimulation. Capacitance measurements were performed with a patch clamp amplifier (AxoPatch 200A; Axon Instruments, Union City, CA, USA) as described previously (Kasai et al. 1996) or with the ‘sine + dc’ method for an EPC-9 amplifier (HEKA, Lambrecht, Germany).

Figure 8. Simultaneous whole-cell measurement of FM1-43 fluorescence and membrane capacitance in PC12 cells.

Figure 8

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A–F, time courses of membrane staining with FM1-43 (A and D), membrane capacitance (B and E), and endocytosis (C and F). Endocytosis was estimated by subtracting the fluorescence traces in A and D from the capacitance traces in B and E, respectively. The time scale in A–C is expanded by a factor of 15 compared with that in D–F. The increases in membrane capacitance (ΔCm) and FM1-43 fluorescence (ΔF) were expressed as a percentage of the values corresponding to the initial area of the plasma membrane. The [Ca2+]i was increased by photolysis of NPE at the time indicated by the arrows (UV). G, time course of staining of the plasma membrane of a cell by rapid application of FM1-43. τ, time constant. H, correlation between peak rates of the increases in membrane capacitance and FM1-43 fluorescence for the fast phase of exocytosis (0–2 s) in 13 cells. I, amplitudes of endocytosis versus those of exocytosis for the fast (0–2 s, •) and slow (2–20 s, ○) phases in 10 cells.

Photoconversion of DAB and EM analysis

Staining of PC12 cells with FM1-43, and subsequent photoconversion of DAB and electron microscopic analysis were conducted as described previously (Kishimoto et al. 2005). The diameters of DAB-positive vesicles were measured for spherical structures with a diameter of > 30 nm that did not exhibit features of endosomes or lysosomes (Prekeris et al. 1998; de Wit et al. 1999). The density of DAB-positive vesicles (μm−2) was converted into the number of vesicles per cell by multiplying by H4πR3/3, where R represents the mean radius of PC12 cells (6.5 μm) and H is a correction factor (0.545) for the effect of thickness of the section (s= 60 nm); H is equal to s/(s+d), where d is vesicle diameter (50 nm).

Results

Ca2+-dependent exocytosis of small vesicles in PC12 cells

We triggered exocytosis in PC12 cells by photolysis of the caged-Ca2+ compound nitrophenyl-EGTA (NPE), which was loaded into the cells in the form of an acetoxymethyl ester (AM). The rapid increase in the intracellular free Ca2+ concentration ([Ca2+]i) resulting from NPE photolysis was estimated, with the use of the indicator fura-2FF, to be 10–25 μm in magnitude and to persist for > 10 s, as previously described (Nemoto et al. 2004). We performed two-photon excitation imaging in a solution containing FM1-43, a membrane tracer that is fluorescent only in the membrane, and does not permeate the plasma membrane (Betz & Bewick, 1992).

Photolysis of NPE induced a rapid increase in FM1-43 fluorescence that was apparent first in the plasma membrane and then in the cytoplasm (Fig. 1A and B). The possibility that FM1-43 had leaked through the intact plasma membrane into the cytoplasm was ruled out by the fact that the observed cytoplasmic staining was more homogeneous than that apparent in cells whose plasma membrane had been permeabilized with saponin (Fig. 1C). The increase in FM1-43 fluorescence (Fig. 1D) was similar in amplitude (range, 15–60%, 45 ± 8.9%, mean ±s.d., n = 13) and time constant (∼1 s) to the increase in membrane capacitance associated with the fast component of exocytosis (see Fig. 8C and H). The zero level of fluorescence in these experiments was determined immediately before application of FM1-43 to cells.

Figure 1. Rapid exocytosis and endocytosis detected with FM1-43 at the lateral surface of PC12 cells.

Figure 1

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A, sequential FM1-43 fluorescence (F) images from a cell loaded with NPE-AM are shown in frames a1 to a4. Photolysis was induced at a time between frames a1 and a2 (UV). The dye was washed out before frame a4. B, the difference images (ΔF) shown in frames b1 to b3 were obtained by subtracting the resting image (frame a1) in A from frames a1 to a3, respectively. C, FM1-43 fluorescence image of a cell treated with saponin (0.1%). D and E, time courses of the changes in FM1-43 fluorescence for the entire section of the cell shown in A. The zero level of fluorescence was obtained before application of FM1-43 to cells. F, cytoplasmic and plasma membrane regions indicated by red and blue circles, respectively, for the cell shown in A. G, time course of the change in fluorescence in the plasma membrane region (blue line) or in the cytoplasmic region (red line) delineated in F. Fluorescence was normalized by that of the entire section of the cell before photolysis and is expressed as a percentage of control values (Fnormalized). H, spatial distribution of FM1-43 fluorescence (AU, arbitrary units) along the line (inset) across the plasma membrane of the cell shown in A. The traces were obtained from difference images (ΔF) at the indicated times after NPE photolysis, with that for 40 s corresponding to 15 s after washout of FM1-43.

The increase in FM1-43 fluorescence was largely unaffected by washout of the tracer (Fig. 1Bb3 and Aa4), remaining at a level of 98 ± 10% (n = 8) (Fig. 1E), indicating that most of the vesicle membranes inserted into the plasma membrane by exocytosis were subsequently taken up by endocytosis. The increase in FM1-43 fluorescence was also retained after washout when the cells were stimulated with a high concentration (120 mm) of K+ (n = 6, unpublished data). Moreover, measurement of the fluorescence increase around the plasma membrane revealed that it peaked 3–4 s after stimulation (Fig. 1F and G) and then decayed by 30–70% over the next 20 s, even though FM1-43 was still present in the extracellular medium. This decrease in fluorescence at the plasma membrane was accompanied by a reciprocal increase in fluorescence in the cytoplasm (Fig. 1G). The rapid increases in fluorescence were localized to the plasma membrane (Fig. 1H). We could not detect spatial gradients of fluorescence in the cytosol (Fig. 1H), suggesting rapid diffusion of endocytic vesicles in the cytosol. Thus, two-photon excitation imaging with FM1-43 revealed the generation of endocytic vesicles at the plasma membrane and their translocation deep into the cytoplasm within a period of 10 s. These observations illustrate that, unlike one-photon confocal microscopy (Smith & Betz, 1996), there is very little photobleaching in TEP imaging due to diffusion of FM1-43-stained organelles from outside of the focal plane (Kasai et al. 2005).

These features of exocytosis were quite different from those of the large dense-core vesicles (LVs) in PC12 cells (Kishimoto et al. 2005). LVs underwent slower exocytosis with a time constant of ∼7 s, and mostly stayed at the site of exocytosis with their fusion pore in the open conformation.

In order to determine diameters of vesicles responsible for the rapid staining of membrane with FM1-43 (Fig. 1B), the diameter of vesicles was estimated by TEPIQ analysis of ΔV/ΔS (where ΔS and ΔV are vesicle volume and surface area), which is applicable to populations of vesicles in which single exocytic events are not resolved (Kasai et al. 2005). TEPIQ analysis of ΔV/ΔS uses both SRB and FM1-43, and we chose to study regions of the cell facing the intercellular space in PC12 cell clusters, where background fluorescence of SRB was small due to the narrow intercellular space (see Fig. 7). We observed both discrete and diffuse increases in SRB fluorescence at the plasma membrane (Fig. 2A). Most of the discrete SRB spots were washed out after removal of SRB (Fig. 2D), similar to spots observed at the cell base (Fig. 1A of Kishimoto et al. 2005), suggestive of LV exocytosis. SRB also diffusely stained the plasma membrane and cytoplasm similarly in spatial distribution to FM1-43 (Fig. 2A and B). Also, as was the case for the FM1-43 signal, the SRB signal often peaked 3–4 s after stimulation, and decayed thereafter (see Figs 5D and 6C). Both dyes were retained after washout of the tracers (Fig. 2D and E). SRB thus appeared to label the same population of vesicles that was diffusely stained by FM1-43.

Figure 7. Ultrastructural identification of endocytic vesicles in PC12 cells.

Figure 7

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Endocytic vesicles were examined by EM in cells loaded with FM1-43. Photoconversion of DAB was induced by FM1-43 molecules remaining after washout. A small amount of FM1-43 molecules were always left in the plasma membrane, and stained the plasma membranes and narrow intercellular spaces (arrows). A, cells immersed in FM1-43 for 20 min at rest. The inset shows a DAB-positive coated vesicle. B–D, cells immersed in FM1-43 for 20 s without (B) or with (C and D) stimulation by photolysis of NPE 10 s after initial exposure to the tracer and fixed 10 s after photolysis. Images were obtained from sites of cell contact (A–C), from deep within the cytoplasm (D). EV, CV, LV, LS, E, and M denote fusion pore-derived small endocytic vesicles, constitutive endocytic vesicles, large dense-core vesicles, lysosomes, endosomes, and mitochondria, respectively. The external scale bar (0.5 μm) applies to all panels with the exception of the inset in A, in which the internal bar represents 50 nm.

Figure 2. Simultaneous imaging of exocytosis with SRB and FM1-43.

Figure 2

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A and B, difference TEP images for SRB (A) and FM1-43 (B) fluorescence measured before and after (averaged between 2 and 10 s after stimulation) NPE photolysis. Arrows indicate discrete exocytic events, whereas boxed regions i and j contain diffuse fluorescence. C, a 6ΔV/ΔS image generated from the data shown in A and B with a pseudocolour coding indicated on the right. D–F, SRB, FM1-43, and 6V/S TEPIQ images of the preparation shown in A to C after washout of the dyes. Images were averaged between 15 and 26 s after washout. Boxed regions p and q in F correspond to boxed regions i and j in A.

Figure 5. Diameters of fusion pores estimated with multicolour TEPIQ analysis.

Figure 5

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A and C, difference images with SRB (left) and 10 kDa FD (right) of cells in clusters loaded with NPE-AM. The original images were obtained before and 10.2 s after photolysis. The boxed region in A is magnified in C. B, D and E, time courses of ΔV based on SRB (ΔVSRB) and on 10 kDa FD (ΔV10 kDx) fluorescence for discrete events (spot b in A), diffuse events at the plasma membrane (box d in C), and cytoplasmic staining (box e in C), respectively. F, difference images with SRB (left) and bis-fura-2 (right) of cells in clusters loaded with NPE-AM. The original images were obtained before and 10 s after photolysis. The asterisk in E denotes slow appearance of 10 kDa FD-accessible vesicles in the cytosol. F, ratio between ΔVdye and ΔVSRB measured at maximal ΔVSRB for discrete events (LV), diffuse events at the plasma membrane (SV), cytoplasmic staining in GTP-γ-S-treated preparations (EV) (Fig. 6D), and constitutive endocytosis (CV). The ratios are plotted against approximated diameters of the dyes. Error bars show s.d. (n = 3–12).

Figure 6. Effects of GTP-γ-S on exocytosis and endocytosis.

Figure 6

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A and B, multicolour TEPIQ analysis of ΔV with SRB and 10 kDa FD for constitutive endocytosis in the absence (A) and in the presence (B) of GTP-γ-S (0.36 mm) in the whole-cell patch-pipette. C and D, multicolour TEPIQ analysis of ΔV diffuse events induced with photolysis of caged Ca2+ at the plasma membrane (C) and cytoplasmic staining (D), respectively, in cells subjected to whole-cell dialysis with (ΔVdye(G)) or without (ΔVdye) GTP-γ-S (0.36 mm).

Ratiometric images of 6ΔV/ΔS (Fig. 2C) revealed that the discrete SRB spots (Fig. 2A) were attributable to vesicles with a diameter of 0.214 ± 0.064 μm (n = 18) (Fig. 3A and D), whereas the diffuse events at the plasma membrane (Fig. 2A) exhibited a diameter of 0.056 ± 0.004 μm (n = 12) (Fig. 3B and E). The time course of the ratio (Fig. 3E) for these latter events indicated that the diameter of the responsible vesicles (SVs) was 0.06 ± 0.004 μm (n = 10) within 0.6 s of the onset of the increase in [Ca2+]i. The rapid generation of small vesicle structures strongly supports that they result from SV exocytosis. The diameter determined is consistent with previous estimates of SV diameters in PC12 cells (Cameron et al. 1991; Linstedt & Kelly, 1991; Bauerfeind et al. 1993; de Wit et al. 1999).

Figure 3. Estimation of vesicle diameter by TEPIQ analysis of ΔV/ΔS.

Figure 3

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A–C, time courses of changes in ΔV and ΔS for a discrete event (the thick arrow in Fig. 2A) (A), diffuse plasma membrane staining (box i in Fig. 2A) (B) and cytoplasmic staining (box j in Fig. 2A) (C). D–F, time courses of changes in 6ΔV/ΔS obtained from the plot shown in A–C. Traces from 5 cells (colour) and their average (black) are superimposed in E.

The diameter of SVs did not appear to change within 2 s (Fig. 3E) when closure of the fusion pore occurred, based on capacitance measurements (see Fig. 8F). The estimated diameter of vesicles apparent at the plasma membrane after washout was 0.055 ± 0.003 μm (n = 12) (Fig. 2F, boxed area p), and that of the endocytic vesicles deep in the cytoplasm (EVs) was 0.056 ± 0.003 μm (n = 9) and 0.057 ± 0.002 μm (n = 9) before (Figs 2C, and 3C and F) and after washout (Fig. 2F, boxed area q; TEPIQ analysis of V/S), respectively. We thus concluded that the diffuse SRB and FM1-43 staining was attributable to SVs and to the subsequently formed endocytic vesicles (EVs).

The diameter of constitutive endocytic vesicles (CVs), which were labelled by immersing cells in dye solution for 10 min without stimulation (Fig. 4A–D), was estimated as 0.090 ± 0.006 μm (n = 7) (Fig. 4E), consistent with the results of EM analysis (see Fig. 7A).

Figure 4. TEPIQ analysis of constitutive endocytic vesicles.

Figure 4

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A and B, difference TEP images with SRB (A) and FM1-43 (B) in a PC12 cell immersed in a solution containing SRB and FM1-43 for 10 min without stimulation. C, 6ΔV/ΔS image. D and E, time courses of changes in ΔV and ΔS (D) and 6ΔV/ΔS (E) obtained from the region r in C.

Mechanisms of endocytosis

To characterize the mechanisms of formation of endocytic vesicles, EVs and CVs, we tested the ability of various fluorescent tracers, used as nanometer-sized probes (Takahashi et al. 2002), to enter and stain vesicles. Cells were immersed in solutions containing both SRB (diameter, 1.4 nm) and one of the other polar tracers and then subjected to NPE photolysis. Discrete spots at the plasma membrane facing intercellular space (Fig. 5A and B) were stained simultaneously with SRB and 10 kDa FD (6 nm), as observed for exocytosis of single LVs at the base of cells (Fig. 6D of Kishimoto et al. 2005). In contrast, regions at the plasma membrane that showed a diffuse increase in SRB fluorescence, reflecting SV exocytosis, were not stained with 10 kDa FD (Fig. 5C and D), indicating that the diameter of the fusion pore remained smaller than 6 nm. We then quantified the ratio between the apparent volume filled with the test tracer (ΔVdye) and that filled with SRB (ΔVSRB) at the time point when ΔVSRB was maximal. Such multicolour ratiometric TEPIQ analysis revealed that the fusion pore of SVs allowed permeation of SRB and pyranine (1.0 nm) but not of bis-fura2 (1.6 nm), Oregon Green 488–BAPTA-2 (2 nm), or 3 kDa FD (4 nm) (Fig. 5F). These observations indicated that EVs were formed at the plasma membrane by ‘direct endocytosis’ reflecting closure of a fusion pore with a diameter of roughly 1.6 nm, without associated flattening of the secretory vesicle into the plasma membrane. Similarly, EVs in the cytosol, which were detected within a short time (< 20 s) after NPE photolysis (Fig. 5E), were selectively stained with SRB but not with 10 kDa FD (Fig. 5E).

In contrast, CVs were also stained with 10 kDa FD (Fig. 6A). The fact that CVs were stained even with 70 kDa FD (diameter, 12 nm) (Fig. 5F) indicates that they were formed from flat plasma membrane, rather than from an existing constricted neck. To examine whether GTPases differentially regulate direct and constitutive endocytosis, we applied guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ-S) to the cell interior by the whole-cell patch-clamp method. GTP-γ-S eliminated the formation of CVs (Fig. 6B), whereas it did not affect rapid Ca2+-induced exocytosis (n = 5) (Fig. 6C), consistent with a previous report in a presynaptic terminal (Takahashi et al. 1999), or the subsequent endocytosis and translocation of EVs into the cytoplasm (n = 5) (Fig. 6D). GTP-γ-S also prevented the appearance of the cytoplasmic vesicles that were stainable with 10 kDa FD (Fig. 6D) and normally evident 20 s after the increase in [Ca2+]i (Fig. 5E, asterisk), indicating that these vesicles were formed by a mechanism similar to that for CVs.

Ultrastructural identification of endocytic vesicles

We confirmed the identity of Ca2+-induced endocytic vesicles revealed by two-photon excitation imaging with FM1-43 by using EM. In these experiments, DAB was photoconverted by FM1-43 (Henkel et al. 1996; Harata et al. 2001; Kishimoto et al. 2005). As a control, we first labelled the constitutive endocytic pathway by immersing cells in FM1-43 for 20 min without stimulation. This treatment resulted in the appearance of many DAB-positive vesicles (CVs) (Fig. 7A) with diameters of 0.04–0.12 μm (0.087 ± 0.046 μm, n = 114), some of which were coated (Fig. 7A, inset). We also detected labelling of coated pits and tubulovesicular structures that resembled early endosomes and lysosomes (Fig. 7A), consistent with the operation of the clathrin-mediated endocytic pathway (Heuser & Reese, 1973; Koenig & Ikeda, 1989). In contrast, cells exposed to FM1-43 for only 20 s before fixation exhibited staining only of the plasma membrane (arrows), and not of endocytic structures (Fig. 7B).

Many small DAB-positive vesicles were detected both adjacent to the plasma membrane and deep within the cytoplasm (Fig. 7C and D) when cells were stimulated by photolysis of NPE during exposure to FM1-43 for 20 s. The cells were fixed 10 s after photolysis, so that the vesicular staining must have occurred within the 10 s after stimulation. None of these vesicles, even those attached to the plasma membrane, were coated, and their diameters ranged between 0.03 and 0.09 μm (0.054 ± 0.010 μm, n = 110). Unexpectedly, many tubulovesicular structures that resembled early endosomes (Prekeris et al. 1998; de Wit et al. 1999) and lysosomes were also stained (Fig. 7C and D), indicating that the endocytic vesicles were transported to these organelles, even to those present deep in the cytoplasm. The number of DAB-positive vesicles ranged between 1 and 4 μm−2, corresponding to a total of 10 000–40 000 vesicles per cell (see Methods) or to 15–62% of the original plasma membrane, consistent with the large increase in surface membrane detected by two-photon excitation imaging with FM1-43 (Fig. 1G) and with measurements of membrane capacitance (see Fig. 8I). The fact that diameters of EVs were similarly estimated with both EM and ΔV/ΔS-TEPIQ analysis supports the conclusion that SRB passively filled in all vesicles that were labelled by FM1-43, and that the TEPIQ diameter is not influenced by merger of endocytic vesicles with endosomes (Kasai et al. 2005).

Interestingly, exocytosis of SVs appeared to be rare at the base of the cells. Structures like EVs were relatively infrequent at the plasma membrane facing the glass cover-slips (see Fig. 7C–E of Kishimoto et al. 2005). This is consistent with the TEPIQ analysis indicating that average diameters of vesicles were always around 0.18 μm (Kishimoto et al. 2005). The reason for the rare occurrence of SV exocytosis at the base of the PC12 cells remains to be clarified.

Simultaneous measurement of membrane capacitance and of FM1-43 fluorescence

Finally, we examined the correlation between our data with FM1-43 and with the membrane capacitance measurement. Changes in membrane capacitance represent the difference between the amount of membrane inserted into the plasma membrane by exocytosis and that removed by endocytosis (Kasai et al. 1996; Smith & Betz, 1996). In contrast, whole-cell FM1-43 fluorescence should not be affected by endocytosis (Smith & Betz, 1996) and therefore provides a measure of cumulative insertion of secretory vesicle membrane into the plasma membrane by exocytosis.

The initial rate of increase in membrane area was similarly revealed by both whole-cell FM1-43 fluorescence (Fig. 8A) and membrane capacitance (Fig. 8B and C), as the maximal slopes of the membrane capacitance and FM1-43 fluorescence correlated tightly in the same cells (Fig. 8H). This indicates that FM1-43 stains most exocytic events, validating our TEPIQ analyses. Due to the rapid insertion of FM1-43 into the plasma membrane, with a time constant of 35 ms (Fig. 8G), there was only a slight delay in the onset of the FM1-43 signal with respect to that of the capacitance increase (Fig. 8C). The time course of the increase in FM1-43 fluorescence was fitted with the sum of two exponential functions with time constants of 0.8 and 10 s, which mainly represent exocytosis of SVs and LVs, respectively, based on TEPIQ analyses (this study and Kishimoto et al. 2005).

We estimated the extent of endocytosis by subtracting the FM1-43 signal (Fig. 8A and D) from the capacitance signal (Fig. 8B and E) (Smith & Betz, 1996). This procedure was possible as both methods estimated the increases in membrane area by percentage changes, allowing direct comparisons of the two measurements. This approach revealed that endocytosis occurred in two phases. The fast and slow phases began with delays of ∼200 ms (Fig. 8C) and ∼10 s (Fig. 8F) after the onset of the increase in [Ca2+]i and developed with time constants of ∼1 and ∼10 s, respectively. The fast phase of endocytosis accounted for most (range, 85–110%; 92 ± 5%, n = 10) of the membrane area added by the fast phase of exocytosis (Fig. 8I), consistent with the fact that most membrane added by this phase of exocytosis was taken up by endocytosis (Fig. 1E). These data also indicated that closure of the fusion pore of SVs occurred within 2 s (Fig. 8F). The slow phase of endocytosis also compensated for slow exocytosis, albeit to a markedly lesser extent (range, 10–250%; 60 ± 20%) (Fig. 8I), consistent with exocytosis and endocytosis of LVs (Kishimoto et al. 2005).

Discussion

With the use of post-fusion labelling of exocytic vesicles with TEP imaging, TEPIQ analysis and EM, we have found massive exocytosis of SVs. The TEPIQ diameters of SVs of ∼55 nm were consistent with the EM analysis. Exocytosis of most SVs were mediated by transient opening of fusion pores, which were followed by translocation of the endocytic vesicles into the cytosol.

Small vesicle exocytosis in non-synaptic preparations

Our data established the existence of Ca2+-dependent exocytosis of SVs in non-synaptic preparations. Similar rapid capacitance increases have been demonstrated in many other cell types including β-cells (Takahashi et al. 1997), chromaffin cells (Ninomiya et al. 1997; Haller et al. 1998), pituitary cells (Rupnik et al. 2000), astrocytes (Kreft et al. 2004), epithelial cells (Ito et al. 1997), mast cells (Kirillova et al. 1993), and fibroblasts (Coorssen et al. 1996; Ninomiya et al. 1996; Kasai, 1999). Since only a small portion of SVs of PC12 cells contain neurotransmitters (Ninomiya et al. 1997), SVs may play housekeeping roles in addition to secretion, such as membrane repair after injury (Steinhardt et al. 1994; McNeil & Steinhardt, 2003) or recycling of membrane proteins (Borgonovo et al. 2002). Although we conclude that SV exocytosis mainly contributes to the fast component of exocytosis in PC12 cells, the contribution of SV exocytosis to distinct kinetic components of exocytosis is probably different among secretory cells, and it should be directly examined in each cell type and each experimental condition. TEPIQ analysis can be utilized for this purpose.

Our data suggest that most SVs are not docked to the plasma membrane prior to a stimulus (Fig. 9), and therefore that pre-stimulus docking is dispensable for Ca2+-dependent exocytosis. This is because our post-fusion labelling of exocytosis revealed enormous increases (45%) in membrane area by exocytosis of SVs, although no accumulation of SVs to the plasma membrane has been reported in undifferentiated PC12 cells. In fact, EM investigations of quick-frozen PC12 cells have demonstrated that docked SVs are so rare that they can account for at most a 3% increase in membrane area (Kasai et al. 1999). Also, synaptophysin immuno-reactive vesicles were scattered throughout the cytosol of PC12 cells (Johnston et al. 1989; Lah & Burry, 1993). The idea that pre-stimulus docking is dispensable for Ca2+-dependent exocytosis is also supported by the fact that deep LVs undergo exocytosis without pre-stimulus docking to the plasma membrane during sequential exocytosis of LVs in PC12 cells (Kishimoto et al. 2005), β-cells (Takahashi et al. 2004) and in acinar cells (Nemoto et al. 2001, 2004).

Figure 9. Pre-stimulus docking and fusion reactions.

Figure 9

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Small vesicles are neither clustered at the plasma membrane nor docked to the plasma membrane prior to stimulation. The fusion pores of SVs are rapidly closed, and vesicles readily detach from the plasma membrane after fusion, because of the absence of pre-stimulus docking. A part of the direct endocytic vesicles are transported to endosomes or lysosomes (the oval). Open bars stands for putative proteins involved in membrane fusions.

Exocytosis of SVs in PC12 cells occurred slowly with a time constant of 1 s. This time constant may be consistent with the fact that post-docking reactions of SVs require 250 ms (Zenisek et al. 2000). The time constant of 1 s is very slow compared with already docked SVs in the active zone of presynaptic terminals, which undergo exocytosis within a fraction of a millisecond. Thus, pre-stimulus docking of SVs to the active zone must be necessary for sub-millisecond exocytosis, which may be enabled by prior complex formation and functioning of SNARE proteins. Pre-stimulus docking, however, is not caused by SNARE proteins (Hunt et al. 1994; Sorensen et al. 2003), and mediated by other proteins, such as Munc-18 (Voets et al. 2001; Korteweg et al. 2005), annexins (Kishimoto et al. 2005) and various active zone proteins (Sudhof, 2004).

Pre-stimulus docking facilitates exocytosis in three different ways. First, it hastens fusion reaction in certain preparations, such as the in the active zone of presynaptic terminals. Second, it promotes the preservation of Ω-shaped profiles of fused vesicles at the site of exocytosis (Fig. 8 of Kishimoto et al. 2005), and facilitates exocytosis by allowing sequential compound exocytosis (Kishimoto et al. 2005). Finally, the preservation of Ω-shaped profiles of fused vesicles at the site of exocytosis has also been proposed for SVs in the active zone, and facilitates exocytosis by enabling rapid reuse of vesicles for subsequent exocytosis (Torri-Tarelli et al. 1985; Murthy & Stevens, 1998; Pyle et al. 2000; Valtorta et al. 2001; Aravanis et al. 2003a). Pre-stimulus docking of SVs is considered to promote the preservation of Ω-shaped profiles of fused SVs, because we found that SVs could readily detach from the plasma membrane after exocytosis in PC12 cells, where there is little pre-stimulus docking of SVs (Fig. 9).

Direct endocytosis

Ceccarelli and colleagues proposed the existence of a ‘direct’ endocytic mechanism for synaptic vesicles distinct from that mediated by coated pits (Ceccarelli et al. 1973; Meldolesi & Ceccarelli, 1981). It has been further hypothesized that endocytic vesicles generated by this direct mechanism rapidly become available for reuse in exocytosis according to a ‘kiss-and-run’ recycling mechanism (Valtorta et al. 2001). The existence of such endocytosis is still controversial (Aravanis et al. 2003b; Gandhi & Stevens, 2003; Rizzoli & Betz, 2004; Yamashita et al. 2005). Moreover, mechanisms of membrane fission for ‘direct’ endocytosis and fates of endocytic vesicles have not been well characterized.

Using a post-fusion labelling approach, we have now demonstrated that endocytic uptake of membrane into the cytoplasm can occur as the irreversible closure of a narrow fusion pore of vesicles (SVs) in PC12 cells. Since we found that SRB, but not bis-fura2, permeated the fusion pore of SVs, it is likely that the diameter of these molecules (1.4 and 1.6 nm, respectively) is the critical factor for such selectivity, given that the results from all six different tracers examined can be explained by their diameters, although possible small contributions of charge or fine structure of the tracer molecules cannot be excluded (see Methods). For instance, the apparent sharp cut off in permeation for bis-fura2 relative to SRB might also be attributed to the presence of fewer negative charges on SRB than on bis-fura2.

The absence of uptake of molecules with a diameter of > 1.6 nm rules out a mechanism similar to that operating for constitutive endocytosis, in which endocytic vesicles are formed from the flat plasma membrane. Indeed, such endocytosis was not affected by GTP-γ-S, suggesting that it does not require GTPases, such as dynamin, which are necessary for constitutive endocytosis. Direct endocytosis may play major role in some synapses (Aravanis et al. 2003b; Gandhi & Stevens, 2003; Staal et al. 2004), but not in neuromuscular junction (Rizzoli & Betz, 2004) and in the calyx of Held nerve terminal (Yamashita et al. 2005).

The absence of uptake of molecules larger than 1.6 nm also indicates that most exocytic SVs in PC12 cells are taken up by direct endocytosis. This property of PC12 cells allowed us to examine the ultrastructural characteristics of EVs in the cytoplasm. Most EVs (> 70%) were rapidly detached from the plasma membrane. Thus, we have, for the first time, shown the existence of running of direct endocytic vesicles, in contrast to ‘kiss-and-stay’ endocytosis reported in many preparations (Burgoyne et al. 2001; Sudhof, 2004; Kishimoto et al. 2005). Moreover, some were even transported to endosomes and lysosomes within 10 s (Fig. 9), indicating that the ‘kiss-and-run’ recycling pathway overlaps with the classical endosomal recycling pathway (de Wit et al. 1999). Unlike SVs in PC12 cells, synaptic vesicles in the central neurones are often reported to stay at the site of exocytosis, and be rapidly reused for subsequent exocytosis. The difference might be ascribed to the presence of pre-stimulus docking of SVs in the active zone of central neurones, but not in PC12 cells.

TEP imaging could possibly resolve individual synaptic vesicle exocytosis in synaptic terminal structure facing the postsynaptic membrane in intact brain preparations. Although we have not succeeded in TEP imaging of exocytosis of individual SVs in PC12 cells because of the background fluorescence, new two-photon compatible tracers have the potential to overcome this hurdle (Albota et al. 1998).

Acknowledgments

We thank T. Kanaseki for helpful suggestions, Y. Miyashita and M. Iino for encouragement, and T. Kise, T. Suzuki and N. Takahashi for technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by research grants from the Human Frontier Science Program Organization, NIH and the Takeda Science Foundation.

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