Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein

INTRODUCTION

Neuroendocrine cells contain large number of dense-core vesicles that are filled with peptide hormones for secretion. Recent studies have suggested that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) (Rothman, 1994; Jahn and Sudhof, 1999) and small GTPase Rabs (Zerial and McBride, 2001) are thought to be required for biogenesis and trafficking of dense-core vesicles. The secretion process (i.e., exocytosis) from neuroendocrine cells of dense-core vesicles consists of 4 steps: 1) the transported dense-core vesicle morphologically attaches to the plasma membrane (tethering), 2) the tethered vesicle forms a tight core SNARE complex at the target plasma membrane (docking), 3) the preparation of exocytosis competent vesicles depending on Mg²⁺-ATP and temperature (priming), and 4) the fusion of the vesicle with the plasma membrane by extracellular stimuli (fusion). Only a small fraction of these vesicles is able to be released rapidly in response to elevated cytosolic Ca²⁺ concentrations, and this fraction is called the readily releasable pool (RRP). The remaining vesicles are thought to constitute a large cytosolic reserve pool awaiting recruitment into the red fluorescent protein (RRP) for exocytosis. However, the molecular machinery involved in preferential release from RRP (i.e., preferential recruitment of dense-core vesicle to the plasma membrane and its exocytosis) remains largely unknown.

Recently, Duncan et al. (2003) revealed, using the vesicle cargo-tagged fluorescent timer protein DsRed-E5 which progressively shifts its fluorescence emission from green to red (Terskikh et al., 2000), that dense-core vesicles segregate into distinct populations based on age in bovine adrenal chromaffin cell. Newly assembled vesicles are immobile and morphologically docked at the plasma membrane shortly after biogenesis, whereas older vesicles are mobile and located deeper in the cells. Furthermore, newly assembled vesicles undergo exocytosis in preference to older vesicles. However, it is not known how the segregation of vesicles according to age is accomplished at the molecular level.

We have recently shown that two small GTPases, Rab3A and Rab27A, are abundantly expressed on the dense-core vesicles and cooperatively regulate the docking step of dense-core vesicle exocytosis in PC12 cells (Tsuboi and Fukuda, 2006a). Furthermore, two Rab3A and Rab27A effector molecules, synaptotagmin-like protein 4-a (Slp4-a, also called granuphilin) and rabphilin, promote dense-core vesicle docking to the plasma membrane (Tsuboi and Fukuda, 2005, 2006b; Tsuboi et al., 2007). Slp4-a and rabphilin share the same domain structures (Cheviet et al., 2004; Fukuda, 2005), but they have opposite effects on exocytosis. Slp4-a has been shown to increase the number of “release-incompetent” vesicles docked to the plasma membrane through interaction with a Munc18-1/syntaxin-1a complex (Gomi et al., 2005; Tsuboi and Fukuda, 2006b; Tomas et al., 2008). In contrast, rabphilin has been shown to increase the number of “release-competent” vesicles docked to the plasma membrane through interaction with synaptosome-associated protein of 25 kDa (SNAP-25) (Tsuboi et al., 2007). These findings lead us to speculate that rabphilin and Slp4-a may regulate segregation of dense-core vesicle recruitment and exocytosis.

In the present study, we investigated the molecular mechanism(s) of preferential dense-core vesicle recruitment and exocytosis in PC12 cells by total internal reflection fluorescence (TIRF) microscopy (Axelrod, 1981) with the vesicle cargo-targeted new fluorescent timer probe monomeric Kusabira Green Orange (mK-GO). Expression of mK-GO–tagged vesicle cargoes in PC12 cells showed that newly assembled vesicles appeared green, those of intermediate age are yellow, and older vesicles are red. Although application of high-KCl stimulation induced newly synthesized dense-core vesicle exocytosis, rabphilin-overexpressing cells showed both newer and older vesicle exocytosis. However, only few exocytotic events were observed in Slp4-a–overexpressing cells. Interestingly, silencing of Slp4-a, by RNA interference-mediated knockdown, resulted in an increase in the number of exocytotic events involving older vesicles. Based on these findings, we propose that Rab27A effectors regulate the preferential release of newly synthesized dense-core vesicle in neuroendocrine cells.

MATERIALS AND METHODS

Protein Expression, In Vitro Spectroscopy, and pH Titrations

Proteins were expressed in Escherichia coli, purified, and characterized spectroscopically as described previously (Nagai et al., 2002). For calculation of molar absorption, coefficients and protein concentrations were measured using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as the standard. pH titrations were performed as described previously (Nagai et al., 2002). To analyze the maturation kinetics from green to red, in vitro translation system, WakoPURE system was carried out with the T7 promoter flanking polymerase chain reaction (PCR) products of “fluorescent timer” version of mK-GO at 37°C for 1 h. The synthesized mK-GO was excited at 509 nm and 560 nm, and the ratio of peak amplitudes of fluorescent intensity were plotted against time.

Site-directed Mutagenesis and Plasmid Construction

To develop the monomeric fluorescent timer mK-GO, we introduced six mutations (K49E/P70V/K185E/K188E/S192D/S196G) into wild-type monomeric Kusabira Orange (mKO) (Karasawa et al., 2004; Tsutsui et al., 2005) by site-directed mutagenesis essentially as described previously (Sawano and Miyawaki, 2000). The other plasmids were prepared as described previously (Tsuboi and Fukuda, 2005, 2006b). DNA constructs such as neuropeptide Y (NPY)-mK-GO and tissue-type plasminogen activator (tPA)-mK-GO reported in this article are distributed with concomitant purchase of cDNA for mK-GO from Medical Biological Laboratory International (Amalgaam, Tokyo, Japan).

Slp4-a and Rabphilin Small Interfering RNA (siRNA) and Immunoblotting

To knockdown endogenous Slp4-a and rabphilin protein expression, we used Stealth RNA interference (RNAi) (Invitrogen, Carlsbad, CA) against mouse Slp4-a mRNA (5′-GGGAACAAAGUGUGCUGUU-3′), mouse rabphilin mRNA (5′-GGAGAUGGUGUGAACCGUU-3′), and matched siRNA negative controls (scramble siRNA). To validate Slp4-a or rabphilin knockdown, 3 μl of Slp4-a, rabphilin, or scramble siRNA stock (20 μM) was transfected into PC12 cells, seeded at 1 × 10⁶ cells per 100-mm dish by using Lipofectamine 2000 (Invitrogen). Two days after transfection, PC12 cells were homogenized in a buffer containing 1 ml of 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 10 μM leupeptin, and 10 μM pepstatin A) in a glass-Teflon Potter homogenizer by 10 strokes at 900-1000 rpm, and the proteins were solubilized with 1% Triton X-100 at 4°C for 1 h. After solubilization with 1% Triton X-100 at 4°C for 1 h, the insoluble material was removed by centrifugation at 15,000 rpm for 10 min. The proteins were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with anti-granuphilin (Slp4-a) goat polyclonal antibody (1/200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-rabphilin goat polyclonal antibody (1/100 dilution; Santa Cruz Biotechnology), and anti-actin mouse monoclonal antibody (1/200 dilution; Millipore Bioscience Research Reagents, Temecula, CA). Immunoreactive bands were visualized with horseradish peroxidase (HRP)-conjugated donkey anti-mouse immunoglobulin (Ig)G (1/5000 dilution; Santa Cruz Biotechnology) or HRP-conjugated rabbit anti-goat IgG (1/5000 dilution; Santa Cruz Biotechnology) and detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

TIRF Microscopy

PC12 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 10% horse serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin, at 37°C under 5% CO₂. For TIRF imaging, PC12 cells were plated onto poly-l-lysine–coated coverslips, and the cells were transfected with 4 μg of NPY-mK-GO or tPA-mK-GO plasmids by using Lipofectamine 2000. To knockdown endogenous Slp4-a or rabphilin protein expression, we cotransfected with 3 μl of Slp4-a or rabphilin siRNA stock (20 μM) and either 3 μg of NPY-mK-GO or tPA-mK-GO vectors into PC12 cells. In contrast, to observe the effect of overexpression of Slp4-a or rabphilin on exocytosis, we cotransfected with 2 μg of pEF-T7-Slp4-a or pEF-T7-rabphilin and either 2 μg of NPY-mK-GO or tPA-mK-GO constructs. The cells were used for experiments in 48 h after transfection. The imaging was performed in a modified Ringer's buffer at 37°C (RB: 130 mM NaCl, 3 mM KCl, 5 mM CaCl₂, 1.5 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4). High-KCl stimulation was achieved by perfusion with 70 mM KCl containing RB (the NaCl concentration was reduced to maintain the osmolarity).

To observe the exocytosis of NPY-mK-GO or tPA-mK-GO at the single vesicle level, we used a dual-color TIRF microscope (Tsuboi et al., 2000, 2004, 2007; Tsuboi and Rutter, 2003; Varadi et al., 2005; Tsuboi and Fukuda, 2006b). We mounted a high numerical aperture objective lens (Plan Apochromatic, 100×, numerical aperture = 1.45, infinity corrected; Olympus, Tokyo, Japan) on an inverted microscope (IX71; Olympus), and we introduced an incident light for total internal reflection illumination through the high numerical aperture objective lens via a single mode optical fiber and two illumination lenses (IX2-RFAEVA-2; Olympus). To excite the mK-GO probes, we used a diode-pumped solid state 488 nm (HPU50100, 20 mW; Furukawa Electronic, Chiba, Japan) and 561 nm laser (85YCA020, 20 mW; Melles Griot, Tokyo, Japan) for total internal fluorescence illumination. For simultaneous imaging of red and green fluorescence, an image splitter (Dual View; Optical Insights, Santa Fe, NM) divided the red and green components of the images with a 565 nm dichroic mirror (Chroma Technology, Rockingham, VT) and passed the red component through a 580 nm long pass filter (Chroma Technology) and the green component through a 500–540 nm bandpass filter (Chroma Technology). The images were then projected side-by-side onto an electron multiplier charge-coupled device (EM-CCD) camera (C9100-02; Hamamatsu Photonics, Hamamatsu, Japan). The laser beams were passed through an electromagnetically driven shutter (VMM-D3J; Unibritz, Rochester, NY), and the shutter was opened synchronously with EM-CCD camera exposure controlled by MetaMorph software version 7.5 (Molecular Devices, Sunnyvale, CA). Images were acquired every 300 ms or otherwise as indicated.

To analyze the TIRF imaging data, single exocytotic events were selected manually, and the average fluorescence intensity of an individual vesicles in a 0.7- × 0.7-μm square placed over the vesicle center was calculated (Tsuboi and Fukuda, 2006a,b, 2007; Tsuboi et al., 2007). To distinguish between fusion events and vesicle retreated, we focused on fluorescence changes just before the disappearance of fluorescent signals. When there was a fusion event, a rapid transient increase in fluorescence intensity (to a peak intensity 1.5 times greater than the original fluorescence intensity within 1 s) was observed, whereas when vesicles moved, the fluorescence intensity gradually decreased to the background level as described previously (Tsuboi and Fukuda, 2006b). The number of fusion events during a 5-min period was counted manually. Data are reported as means ± SE of at least 25 individual experiments. Statistical significance and differences between means were compared by one-way analysis of variance followed by the Newman–Keuls multiple comparison test with Prism software (GraphPad Software, San Diego, CA).

RESULTS

Biochemical and Spectral Properties of Fluorescent Timer mK-GO

We previously generated a monomeric version of Kusabira Orange, mKO, from dimer version of wild-type Kusabira Orange fluorescent protein (KO) by introducing mutations in binding surface (Karasawa et al., 2004). During the process of developing brighter and more soluble mutant of mKO, we found a mutant mKO displaying an intermediate green fluorescence transiently in the course of chromophore maturation. We therefore performed additional mutagenesis and finally obtained a color change version (i.e., fluorescent timer) of mKO molecule. In the mKO-base timer fluorescent protein, six mutations (K49E, P70V, K185E, K188E, S192D, and S196G) were eventually introduced into mKO. Then, we named this molecule as a monomeric fluorescent timer version of Kusabira Green Orange or mK-GO.

We analyzed the spectral properties of purified recombinant mK-GO protein prepared from bacteria. The absorption spectrum of mK-GO at pH 7.4 displayed two peaks, one peak at 500 nm (ε = 35,900 M⁻¹ cm⁻¹) and one peak at 548 nm (ε = 42,000 M⁻¹ cm⁻¹) at pH 7.4 (Figure 1A). The absorption spectrum of mK-GO was slightly and modestly pH-sensitive; the apparent pK_a was determined to be ∼6.0 and 4.8 at absorbance of 500 nm and 548 nm, respectively (Figure 1B). Excitation and emission spectra of mK-GO are shown in Figure 1C. After excitation at 500 nm or 548 nm, the protein emitted a green or red, peaking at 509 nm or 561 nm, respectively. We next determined the time course of fluorescence maturation in synthesized mK-GO protein by in vitro translation (Figure 1D). The ratio of orange per green fluorescence was lineally increased and reached at plateau at ∼10 h. The time for the half-maximal fluorescence development is ∼6 h which is faster than DsRed-based fluorescent timer (Terskikh et al., 2002). Together, these results suggest that mK-GO is a monomeric version of fluorescent timer that is suitable for monitoring the age of the targeting protein.

NPY- and tPA-mK-GO Overexpressing PC12 Cells Show Punctate Structure with Green and Red Florescence

To examine the relationship between dynamics of the dense-core vesicle exocytosis and its age, we first generated DsRed-E5-tagged neuropeptide Y (NPY-timer) (Terskikh et al., 2000). Although previous study has shown that DsRed-E5–tagged atrial natriuretic factor (ANF-timer) were targeted to the dense-core vesicles in bovine adrenal chromaffin cells (Duncan et al., 2003), NPY-timer in PC12 cells were localized in both cytosol and vesicles with some aggregates (Figure 2A). The most likely explanation for the low targeting efficiency of NPY-timer in PC12 cells is that the fluorescent timer protein (i.e., DsRed-E5) is a tetramer and therefore could form aggregates through cross-linking with the cargo protein or other proteins when it is fused to the dense-core vesicle cargo proteins (i.e., NPY). When our newly developed monomeric fluorescent timer protein (mK-GO)-tagged NPY and tPA were expressed in PC12 cells, the fluorescence was localized to vesicular structures (Figure 2, B and C) consistent with their reported association with the dense-core vesicle. Moreover, the mK-GO-tagged vesicles in 48 h after transfection showed green and red fluorescence, and the percentage of the newly synthesized green vesicles, intermediate yellow vesicles (green and red), and older red vesicles were ∼20, 70, and 10%, respectively (Figure 2D). We also observed the time-dependent change in vesicle color from green to red in live PC12 cells at the similar time course as in vitro-translated mK-GO (Figure 1 and Supplemental Figures 1 and 2). Therefore, these results suggest that the fusion proteins with the monomeric timer protein, mK-GO do not disturb proper targeting to the dense-core vesicles and retain color timer property to segregate the dense-core vesicles according to its age.

NPY and tPA Are Preferentially Released from Newly Synthesized Dense-Core Vesicles in PC12 Cells

Dense-core vesicles showed green, yellow, and red fluorescence and each florescence was implicated as its age: new, intermediate, and old, respectively. To analyze the single vesicle release behavior related to its age, we monitored the dynamics of single exocytotic events within ∼100 nm beneath the plasma membrane by TIRF microscopy. High-KCl stimulation (70 mM) caused newly synthesized NPY-mK-GO-labeled vesicles (i.e., green vesicles) to brighten and suddenly spread during release of the fluorescent peptide, with an identical time course in older (i.e., yellow or red) vesicles (Figure 3, A–F). Consistently, tPA-mK-GO–labeled green, yellow, and red vesicles showed similar exocytotic dynamics (data not shown). These data indicated that overexpression of NPY- or tPA-mK-GO in the PC12 cells did not disturb their normal secretory behavior and that the kinetics of new and old vesicle exocytosis were identical.

Because a previous study has shown that the newly assembled dense-core vesicle cargo ANF is preferentially released from bovine adrenal chromaffin cells, we next investigated whether newly synthesized dense-core vesicle cargoes NPY and tPA were also preferentially released from the PC12 cells. To examine preferential vesicle release, we counted the total number of NPY- or tPA-mK-GO release events (i.e., green-, yellow-, and red-colored vesicle exocytosis) during high-KCl stimulation. The mean number of plasma membrane-docked NPY-mK-GO–labeled dense-core vesicles (125 ± 38 vesicles per cells, n = 31 cells) was not significantly different from that in cells labeled with tPA-mK-GO (138 ± 25 vesicles per cells, n = 28 cells) (Figure 3G), and the ratio of membrane-docked NPY green, yellow, and red vesicles was indistinguishable from that in tPA vesicles (Figure 2D). However, under high-KCl stimulation, very few exocytotic events involving plasma membrane-docked red colored-NPY-mK-GO vesicles were observed (Figure 3, G and H). Similarly, with tPA-mK-GO, which has a larger molecular size than NPY, exocytotic responses were observed mainly from plasma membrane-docked green- and yellow-colored vesicles (Figure 3, G and H). These results suggest that newly synthesized dense-core vesicle cargoes are released preferentially in PC12 cells.

Overexpression or Depletion of Rabphilin Disturbed the Preferential Exocytosis on Its Age

We have demonstrated previously that both overexpression of rabphilin and Slp4-a greatly increased the number of plasma membrane-docked vesicles. Overexpression of rabphilin facilitated high-KCl-induced NPY-Venus release (Tsuboi and Fukuda, 2005; Tsuboi et al., 2007), whereas overexpression of Slp4-a did not (Tsuboi and Fukuda, 2006b). These data indicated that both rabphilin and Slp4-a promote the docking of dense-core vesicles to the plasma membrane, but the function of those proteins on the dense-core vesicle exocytosis is completely opposite. We therefore used NPY-mK-GO probe to elucidate the functional difference between rabphilin and Slp4-a on the dense-core vesicle exocytosis.

Overexpression of rabphilin increased plasma membrane-docked vesicles (Figure 4, A and D), especially red vesicles (Figure 4B), whereas knockdown of endogenous rabphilin by siRNA (Figure 4C) decreased the number of plasma membrane-docked vesicles revealed by TIRF microscope (Figure 4, A and D). However, it is possible that overexpression of rabphilin affects the distribution of green- and red-colored vesicles inside of the cells. To check this possibility, we observed the vesicle color under the confocal microscope. As we expected, no differences in the population of green- and red-colored vesicle were observed in the overexpression or depletion of rabphilin (Supplemental Figure 3, A–C).

A previous report demonstrated that newly assembled vesicles were immobile at the plasma membrane and older vesicles were located deeper in the cell (Duncan et al., 2003). Therefore, the overexpression of rabphilin seems to enhance the transport of old red vesicles located deeper in the cell to the plasma membrane. In contrast, no significant differences between the ratio of plasma membrane-docked vesicles of each color in control cells and in rabphilin-depleted cells (Figure 4B) revealed that depletion of endogenous rabphilin decreased the number of plasma membrane-docked vesicles of all colors (Figure 4E). These data suggest that the plasma membrane-docked vesicles were bound to rabphilin for transport regardless of its age; therefore, the decreased number of vesicles on the plasma membrane by depletion of endogenous rabphilin seems to be unrelated to vesicle age. We also found that exocytotic responses occurred mainly from plasma membrane-docked green- and yellow-colored vesicles in NPY-mK-GO–expressing control cells. Interestingly, overexpression of rabphilin increased the exocytotic response from yellow- and red-colored vesicles but not green-colored vesicles, whereas depletion of endogenous rabphilin inhibited the exocytotic response from all-colored vesicles (Figure 4F). These data suggest that overexpression or depletion of rabphilin inhibited the preferential newly synthesized dense-core vesicle exocytosis. Our results also indicated that plasma membrane-docked vesicles in rabphilin-overexpressed cells are more readily released than those in rabphilin-depleted cells (Figure 4F). Previous studies suggested that exocytosis occurred at specific sites on the plasma membrane because of the inhomogeneous distribution of SNARE proteins (i.e., syntaxin1-a) (Ohara-Imaizumi et al., 2007). Therefore, the dense-core vesicles in rabphilin-depleted cells might not be recruited to the right place on the plasma membrane where the exocytosis machinery exists. Conversely, the newly synthesized green vesicles in rabphilin-overexpressed cells might not always be existed in the right exocytotic place because yellow or red old vesicles have already been there. Together, these findings suggest that rabphilin regulates the transport of newly synthesized dense-core vesicles to right place on the plasma membrane.

Silencing of Slp4-a Increased the Number of Older Vesicle Exocytosis

We next observed the effect of overexpression or depletion of Slp4-a on the age-dependent preferential exocytosis. Overexpression of Slp4-a significantly increased the number of plasma membrane-docked NPY-mK-GO vesicles (Figure 5, A and D), whereas depletion of Slp4-a expression by siRNA (Figure 5C) had the opposite effect (Figure 5, A and D), suggesting that Slp4-a promotes the docking step in exocytosis. Interestingly, overexpression of Slp4-a increased the number of plasma membrane-docked red-colored NPY-mK-GO vesicles (Figure 5, A, Slp4-a panels, and B), whereas Slp4-a siRNA-transfected cells had increased numbers of plasma membrane-docked green-colored NPY-mK-GO vesicles (Figure 5, A, Slp4-a-siRNA panels, and B). Because Slp4-a inhibits the fusion step in exocytosis, unreleased vesicles stayed on the plasma membrane. Subsequently, the vesicles get older and become red-colored. In contrast, the depletion of endogenous Slp4-a promotes the fusion step on exocytosis; therefore, only newly assembled green-colored vesicles are present on the plasma membrane (Figure 5E). Meanwhile, consistent with overexpression and depletion of rabphilin, no differences in the population of green and red-colored vesicle were observed in the overexpression or depletion of Slp4-a (Supplemental Figure 3, D and E).

NPY-mK-GO vesicle releasing events, involving vesicles of all colors, were decreased in Slp4-a overexpressing cells (Figure 5F), suggesting that exogenous Slp4-a was bound to both new and old vesicles, and inhibited the fusion of all colored secretory vesicles. In contrast, the release of old red vesicles were increased in Slp4-a depleted PC12 cells (Figure 5F), indicating that endogenous Slp4-a was bound to older vesicles to inhibit the fusion of secretory vesicles. Taken together, these findings suggest that Slp4-a regulates age-dependent preferential exocytosis by inhibiting the release of old vesicles.

DISCUSSION

In the present study, we demonstrate that NPY- or tPA-mK-GO is functionally targeted to the dense-core vesicles in PC12 cells, whereas NPY-timer (i.e., DsRed-E5) is not (Figure 2). Previous reports have shown that the florescent timer protein fused to ANF-timer was functionally targeted to secretory vesicles in bovine adrenal chromaffin cells and that the vesicles carrying ANF-timer were exocytosed functionally by high-KCl stimulation (Duncan et al., 2003). The difference of targeting efficiency between our present data and previous reports could result from the distinct character of the cells and/or secretory vesicles derived from different cell lines. Generally, the dense-core vesicles store and release chromogranins and secretogranins (also known as “granins”), a unique group of acidic, soluble secretory proteins (O'Connor and Frigon, 1984). Several granins undergo aggregation induced by low pH and high calcium levels and interact with other components of the matrix of the dense-core vesicles, such as catecholamines, serotonin, and histamine (Kim et al., 2001). Because pH and calcium concentrations in the Golgi complex varies depending on the cell type (Dannies, 2002), the tetrameric fluorescent timer protein might produce aggregates in PC12 cells, but not in bovine adrenal chromaffin cells. As a result, the aberrant folding of timer fluorescent protein disturbs the proper processing of the proNPY and/or sorting at the trans-Golgi network into maturing secretory vesicles in PC12 cells (Molinete et al., 2000).

How can the preferential recruitment of rabphilin to newly synthesized vesicles be established? It has been shown that Rab3A and Rab27A are recruited to immature vesicles shortly after budding and are associated preferentially with newly synthesized vesicles (Handley et al., 2007). Because rabphilin is bound to Rab3A and Rab27A as an effector, rabphilin consequently might be recruited to newly synthesized vesicles via Rab3A and Rab27A. However, it has been also shown that rabphilin transiently associates with the actin cytoskeleton via the actin-binding protein α-actinin (Kato et al., 1996; Baldini et al., 2005) and rapidly exchanges between the dense-core vesicles and cytosol (Handley and Burgoyne, 2008). Therefore, the association of rabphilin to Rab3A and/or Rab27a is not sufficient to explain the mechanism of rabphilin recruitment to the newly synthesized vesicles. Another explanation is that vesicles lose the binding ability to rabphilin in a time-dependent manner. Although several molecules are bound to vesicles to regulate exocytosis, vesicles have a limited number of rabphilin binding sites (i.e., Rab3A and Rab27A) on their surfaces. The newly synthesized vesicles have more rabphilin binding sites than the old vesicles because new vesicles have fewer chances to encounter other vesicle-binding molecules. After all, rabphilin is recruited to newly synthesized vesicles, and facilitates the transport of the new vesicles to the plasma membrane to promote age-dependent preferential exocytosis.

Knockdown of endogenous Slp4-a by specific siRNA decreased the number of plasma membrane-docked NPY- or tPA-mK-GO vesicles and induced exocytosis from both newer and older NPY- and tPA-mK-GO vesicles (Figure 5). Interestingly, Slp4-a–deficient mice whose pancreatic β cells contained a decreased number of attached vesicles on the plasma membrane, exhibited increased insulin secretion activity (Gomi et al., 2005; Kasai et al., 2008). Thus, it is likely that the mechanism of preferential exocytosis by vesicle age is conserved in insulin secretion on pancreatic β cells as well as in other neuroendocrine cells. Previous studies have also shown that chronic exposure of pancreatic β cells to high glucose decreased the expression of granuphilin/Slp4 (Abderrahmani et al., 2006), and that granuphilin/Slp4 is involved in impaired insulin secretion in diabetic mice (Kato et al., 2006). Accordingly, preferential exocytosis is a potential therapeutic target for treatment of diabetes mellitus.

Based on our present findings, we propose following model for preferential exocytosis in neuroendocrine cells (Figure 6): 1) rabphilin is targeted to Rab27A on the newly synthesized dense-core vesicles; 2) rabphilin-attached dense-core vesicle is then transported to the plasma membrane; and 3) incidentally, rabphilin directly binds to SNAP-25 in the plasma membrane. The resulting Rab27A–rabphilin–SNAP-25 complex links the dense-core vesicles to the plasma membrane and enhances exocytotic events (Tsuboi and Fukuda, 2005). This route presumably corresponds to the “readily releasable pool” for the dense-core vesicle exocytosis. However, in some cases, rabphilin-attached dense-core vesicles cannot bind to SNAP-25. This vesicle will follow different route: 4) rabphilin detaches from Rab27A on the middle-aged dense-core vesicles; 5) Slp4-a, other Rab27A effector, occupies Rab27A on the dense-core vesicles, instead of rabphilin; and 6) Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with closed-form syntaxin-1a, which does not contribute to SNARE complex formation, on the plasma membrane. The resulting quadripartite protein complex (i.e., Rab27A-Slp4-a-Munc18-1-syntaxin-1a) forms a tight complex between the dense-core vesicle and plasma membrane and inhibits high-KCl-induced exocytosis (Tsuboi and Fukuda, 2006b). This route presumably corresponds to the “reserve pool” for the dense-core vesicle exocytosis. Because the vesicle could not detach from the plasma membrane, the vesicles bound to Slp4-a subsequently become old. Although disassembly of the quadripartite protein complex must be necessary for the dense-core vesicle exocytosis, when and how the quadripartite protein complex disassembles remains completely unknown (Figure 6).

In summary, we demonstrate by live cell TIRF imaging combined with the use of new fluorescent timer probes that Slp4-a and rabphilin regulate the segregation of dense-core vesicles according to vesicle age. We propose that rabphilin works as a molecular filter for segregation of readily releasable pool from reserve pool and Slp4-a as a molecular filter for segregation of reserve pool from readily releasable pool.

FOOTNOTES

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