Article |
Address correspondence to François Darchen, Institut de Biologie Physico-Chimique, CNRS UPR 1929, 13 rue Pierre et Marie Curie, 75005 Paris, France. Tel.: 33-1-58-41-50-85. Fax: 33-1-58-41-50-23. email: Francois.Darchen{at}ibpc.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Rab27A; MyRIP; exocytosis; actin; neuroendocrine cell
The online version of this article includes supplemental material.
Abbreviations used in this paper: 5-HT, 5-hydroxytryptamine; CTL, cytotoxic T lymphocyte; EW-FM, evanescent wave fluorescence microscopy; hGH, human growth hormone; MSD, mean square displacement; NPY, neuropeptide Y; SERT, serotonin transporter; SG, secretory granule.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A similar actin-dependent capture of the pigment-producing organelles, termed melanosomes, has been described in skin melanocytes (Wu et al., 1998), and a detailed molecular mechanism has been recently provided. The GTP-binding protein Rab27A, which is associated with the membrane of melanosomes, recruits melanophilin, the product of MLPH, which in turn recruits myosin-VA (Fukuda et al., 2002b; Hume et al., 2002; Strom et al., 2002; Wu et al., 2002). In humans, mutations in RAB27A cause Griscelli syndrome, an autosomal recessive disorder characterized by partial albinism and uncontrolled T lymphocyte and macrophage activation (hemophagocytic syndrome) (Ménasché et al., 2000). In melanocytes isolated from these patients or from ashen mice bearing loss-of-function mutations in Rab27A, melanosomes are concentrated in the perinuclear region and cannot accumulate at the distal ends of the dendritic extensions (Wilson et al., 2000; Bahadoran et al., 2001; Hume et al., 2001; Wu et al., 2001). Consequently, melanin cannot be transferred to keratinocytes. Similar impairment of melanosome transport is observed in leaden and dilute mice bearing mutations in MLPH (Matesic et al., 2001) and in MYO5a (Strobel et al., 1990; Provance et al., 1996), respectively. These observations indicate that Rab27A, melanophilin, and myosin-VA mediate the physical link between melanosomes and F-actin.
In retinal pigment epithelial cells, another melanosome-associated complex made of Rab27A, MyRIP, and myosin-VIIA was described recently (El-Amraoui et al., 2002). MyRIP was found to have a broad tissular distribution (Fukuda and Kuroda, 2002), suggesting that its function may not be restricted to melanosome trafficking. In particular, MyRIP is expressed in the retinal synaptic region and, upon expression in pheochromocytoma PC12 cells, was targeted to the tip of neurites that were enriched in SGs. These observations suggested that Rab27A and MyRIP could have a role in secretory vesicle trafficking. Consistently, the activity of Griscelli and ashen cytotoxic T lymphocytes (CTLs) is reduced due to a defect in lytic granule secretion (Ménasché et al., 2000; Haddad et al., 2001; Stinchcombe et al., 2001). Moreover, Rab27A is associated with insulin-containing granules (Yi et al., 2002).
Here we report that Rab27A and MyRIP are associated with large dense core granules in adrenal chromaffin cells and pheochromocytoma PC12 cells and control the secretory activity in a manner that depends on the state of the actin cortex. Moreover, they reduce the mobility of SGs beneath the plasma membrane. The results are consistent with Rab27A and MyRIP bridging vesicles to F-actin and regulating the movement of vesicles within the actin cortex.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The subcellular localization of Rab27A was investigated by cell fractionation of chromaffin cells. The protein was hardly detectable in the cytosol. A crude membrane fraction was separated on a sucrose gradient. Rab27A, the vesicular monoamine transporter VMAT2 and the v-SNARE protein VAMP2 were enriched in the same dense fractions of the gradient, consistent with an association of Rab27A with SGs (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1). In contrast, the distribution of Rab27A was not similar to that of lysosomal ß-glucuronidase. MyRIP was detected in chromaffin and PC12 cell extracts as a single band (96 kD) but was not detected in gradient fractions, presumably because its membrane association is rather labile. Next, the localization of Rab27A and MyRIP was studied by confocal fluorescence microscopy. Chromaffin cells labeled with anti-Rab27A antibodies displayed a punctated labeling distributed throughout the cytoplasm. Moreover, most of the Rab27A-positive structures were also decorated by an antiserum raised against chromogranin A/B, a component of SG matrix (Fig. 1, AC), and by an antidopamine ß-hydroxylase, a marker of SG membrane (not depicted). The distribution of Rab27A was also analyzed in NGF-differentiated PC12 cells. As illustrated in Fig. 1 (DF), the overall distribution of Rab27A was very similar to that of SGs (stained by an antichromogranin A/B antiserum), with a marked enrichment at the tip of neurites. Also, GFP-tagged Rab27A was expressed in PC12 cells, and its localization was very similar to that of chromogranin B, stained with a monoclonal antibody (Fig. 1, GI). In contrast, LYAAT, an amino acid lysosomal transporter (Sagné et al., 2001), displayed a very different intracellular distribution (Fig. 1, JL). Together these results indicate that Rab27A is associated with SGs.
|
|
|
|
Rab GTPases switch between active GTP-bound and inactive GDP-bound conformations. To investigate the function of Rab27A in regulated secretion, wild-type Rab27A or mutants defective in GTP binding (T23N) or in GTPase activity (Q78L) were transiently expressed in PC12 cells. Rab27A-T23N was supposed to interfere with the function of endogenous protein by competing for a guanine nucleotide exchange factor that catalyzes the exchange of GTP for GDP. Rab27A-Q78L was used to increase levels of active GTP-bound Rab27A. Overexpressed Rab27A and Rab27A-Q78L displayed a punctated distribution very similar to that of endogenous Rab27A (Fig. 1, GI; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1). In contrast, Rab27A-T23N gave a diffuse staining of the cytosol (Fig. S2). Rab27A-T23N did not induce any significant change of secretory activity. In contrast, GTPase-deficient Rab27A-Q78L and, to a less extent, wild-type Rab27A have a pronounced inhibitory effect on secretion (Fig. 5 A). Similar effects of Rab27A proteins were observed in cells permeabilized with -toxin or streptolysin-O (Rab27A WT: -24 ± 6.5%, mean ± SEM, n = 5 experiments; Rab27A-Q78L: -57 ± 4.6%, mean ± SEM, n = 3 experiments). As these pore-forming toxins bypass voltage-dependent calcium channels, the inhibitory effect of Rab27A was not due to reduced Ca2+ influx. Rab27A-Q78L inhibited secretory responses over a wide range of free calcium concentrations without apparent shift in the calcium sensitivity of the secretory apparatus (unpublished data). The effect of Rab27A-Q78L was compared with that of other GTPase-deficient Rab proteins. Rab3A-Q81L inhibited secretion as previously reported (Johannes et al., 1994; Schonn et al., 2003), but GFP-tagged Rab8, Rab11, and Rab13 did not interfere with the secretory activity of PC12 cells (Fig. 5 B), although they were properly expressed (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1). Thus, the effect of Rab27A on exocytosis is specific.
|
|
|
|
Secretory effects of Rab27A and MyRIP depend on the state of actin cortex
Biochemical data are consistent with a role for Rab27A and MyRIP in linking vesicles to F-actin. Entrapment of SGs within the actin cortex might thus be responsible for their inhibitory effect on secretory responses. To test this hypothesis, the effect of Rab27A and MyRIP on secretory responses was measured in cells treated with drugs that interfere with actin polymerization. Latrunculins favor actin depolymerization (Spector et al., 1983; Lang et al., 2000), whereas jasplakinolide promotes actin polymerization (Fig. 9 A). Under the conditions used, latrunculin B increased the secretory activity, while jasplakinolide had no significant effect on secretion. In latrunculin Btreated cells, the inhibitory effect of Rab27A-Q78L was significantly reduced compared with the one observed in the absence of drug (Fig. 9 B). Latrunculin B also reduced the effect of MyRIP. On the other hand, the inhibition of secretion induced by MyRIP overexpression was much stronger after jasplakinolide treatment than in standard conditions (Fig. 9 B). This effect is probably mediated by direct interaction of MyRIP with F-actin because it was not observed with MyRIP-Cter. The effect of granuphilin, another putative effector of Rab27A, was not increased by jasplakinolide (Fig. 9 B). In contrast to drugs that act on the actin cortex, microtubule depolymerization by nocodazole did not modify the effect of Rab27A and MyRIP (not depicted). The inhibitory effect of Rab27A and MyRIP, which does not by itself modify the actin cortex (not depicted), is thus partly dependent on the integrity and dynamics of the cytoskeleton.
|
|
Rab27A-Q78L or various MyRIP constructs were transiently expressed in PC12 cells together with NPYGFP (see Videos 15 for representative examples, available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1). All SGs that could be followed for >15 s were tracked (150700 SGs from 1560 cells per group). Expression of Rab27A-Q78L or MyRIP clearly shifted the distribution of Dx,y toward lower values (not depicted) and lowered the mean diffusion coefficient (Fig. 10 E), indicating reduced mobility of SGs. In contrast, expression of MyRIP-RBD and MyRIP-Cter increased the mean diffusion coefficient. None of these proteins affected the mean number of SGs per area unit (unpublished data). In latrunculin-treated cells, SG mobility was enhanced but was insensitive to MyRIP overexpression (Fig. 10 E, right).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In agreement with the localization of Rab27A on SGs, the Rab27A-binding protein MyRIP colocalized with Rab27A and SG markers. The granular localization of MyRIP depends on its NH2-terminal Rab27A-binding domain because truncated constructs lacking this domain are mislocalized (El-Amraoui et al., 2002; Fig. 4 C).
Studies on Griscelli syndrome or ashen CTLs lacking Rab27A revealed a defect in lytic granule exocytosis, suggesting that Rab27A is an essential component of the secretory machinery. During completion of this study, Fukuda et al. (2002a) reported that overexpression of wild-type Rab27A increased the secretory activity of PC12 cells. Similar effects of Rab27A on insulin secretion were also reported recently (Yi et al., 2002). In chromaffin and PC12 cells, we found by several approaches that Rab27A and, more especially, GTPase-deficient Rab27A (that is properly targeted to SGs; Fig. S2) reduced the magnitude of the secretory response (Figs. 59). Rab27A-Q78L reduced not only the amount of released secretory products but also the frequency of release events, as measured by carbon fiber amperometry. This apparent discrepancy might result from differences in assays used to measure secretion, from the relative importance of various Rab27A effectors in different cells, or from a rate-limiting effect of GTP hydrolysis by Rab27A. An increased probability of spontaneous fusion does not seem to account for the observed inhibition of stimulus-dependent release by Rab27A and Rab27A-Q78L, as was proposed recently by Schlüter et al. (2002) for Rab3A, another GTPase associated with SGs. Indeed, we did not detect any significant increase in release activity in the resting state.
Our observations suggest that MyRIP mediates, at least partly, the effect of Rab27A on secretion and provides a physical link between SGs and actin. Indeed, both proteins reduced the "sustained" component of release that involves recruitment of vesicles from the "reserve" pool. Moreover, both Rab27A- and MyRIP-induced effects on secretion were modulated by drugs that affect the actin cortex. Latrunculin B, which reduces the thickness of the actin cortex, also reduced the inhibitory effects of Rab27A-Q78L and MyRIP, while the actin-stabilizing drug jasplakinolide strongly strengthened the effect of MyRIP (Fig. 9). Finally, both MyRIP and Rab27A-Q78L restrict the motion of SGs in the actin-rich region of the cell imaged by EW-FM, whereas MyRIP-RBD and MyRIP-Cter, which are supposed to reduce the association of endogenous MyRIP with Rab27A, enhanced the mobility of SGs (Fig. 10 E). Latrunculin was found to increase SG mobility in mock-transfected cells, in agreement with F-actin acting as a barrier, and to prevent the effect of MyRIP on SG motion, in agreement with MyRIP tethering SGs to F-actin.
Previous studies revealed that SGs are captured at the cell periphery by a process involving both microfilaments and microtubules (Rudolf et al., 2001). MyRIP and Rab27A may play a role in SG capture similar to that of Rab27A/melanophilin/myosin-Va in the actin-dependent retention of melanosomes. However, if actin filaments may favor the retention of SGs at the cell periphery, they also hamper the diffusion of SGs toward the plasma membrane, as indicated by the increase in SG mobility and release induced by latrunculin. MyRIP may link SGs to actin via myosin-Va, which was recently shown to favor the dispersion of immature SGs within the actin cortex (Rudolf et al., 2003) and to be involved in secretion (Rosé et al. 2003). Recruitment of a molecular motor might indeed promote the motion of SGs within the actin cortex. MyRIP may also link SGs to actin filaments via a myosin-independent mechanism involving the COOH-terminal region of MyRIP (Fig. 4; Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1). This region displays significant sequence similarity with the actin-binding domain of melanophilin, suggesting that MyRIP may also bind F-actin directly. Our data indicate that this myosin-independent binding of MyRIP to actin is responsible for the restricted mobility of SGs. Indeed, SG motion was reduced by MyRIP but increased by MyRIP-Cter, which binds to myosin-Va but not to actin (Fig. 4). The motion of SGs within the actin cortex thus depends on the dynamics of actin remodeling and of Rab27/MyRIP/actin interaction.
The inhibition of secretion observed upon expression of Rab27A or MyRIP is apparently correlated with the restricted motion of SGs within the actin cortex. In the case of MyRIP, both effects display the same dependence on actin organization. Clearly, increasing the degree of actin polymerization (by jasplakinolide) and the interaction of SGs with actin (by MyRIP) is detrimental to the secretory process, most likely by preventing the access of SGs to release sites. However, MyRIP-Cter, which increased SG mobility, also reduced the magnitude of the secretory response. In this case, the effect on secretion was not strengthened by jasplakinolide. This result suggests that MyRIP may interfere with the secretory process via another mechanism. This hypothesis deserves further study. It will also be interesting to investigate the putative function of MyRIP in other secreting cells such as CTLs and neurons.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid encoding rat SERT (pcDNA3-SERT) and NPYGFP were provided by R. Blakely (Vanderbilt University School of Medicine, Nashville, TN) and W. Almers (Oregon Health Sciences University, Portland, OR), respectively. pcDNA3-granuphilin was a gift of R. Regazzi (University of Lausanne, Lausanne, Switzerland). pEGFP-C1 was from BD Biosciences Clontech. Other plasmids encoding MyRIP and Rab27A constructs were described previously (El-Amraoui et al., 2002; Ménasché et al., 2003). Streptolysin-O and -toxin were obtained from S. Bhakdi (Johannes Gutenberg University, Mainz, Germany) (Bhakdi et al., 1993). Latrunculin B and jasplakinolide were from Calbiochem. Other chemicals were purchased from Sigma-Aldrich.
Cell culture and transfection
Primary dissociated chromaffin cells from bovine adrenal medulla were prepared by retrograde collagenase perfusion and cultured as previously described (Darchen et al., 1990). PC12 cells were cultured and transfected as described previously (Schonn et al., 2003). Where indicated, NGF (50 µg/ml) was added to the culture medium. COS-7 cells were cultured in DME supplemented with 10% FBS at 37°C under 5% CO2. Chromaffin and COS-7 cells were transfected by electroporation using Optimix electroporation kit (Equibio). In brief, freshly prepared cells were suspended in Optimix buffer A and collected by centrifugation (800 g, 10 min). Cells (1.5 x 106) were resuspended in 55 µl Optimix buffer B containing vector DNAs (5 µg). A single electric shock (PC12: 650 V/cm, 24 ms; COS: 625 V/cm, 8 x 3 ms) was applied using a PS10 electropulsator (Jouan). Electroporated cells were immediately recovered in warm culture medium and plated onto polylysine-coated glass coverslips. Experiments were generally performed 3 d after transfection.
Immunoelectron microscopy
PC12 and chromaffin cells were fixed with 2% paraformaldehyde in 0.2 M phosphate buffer, pH 7.4, for 1 h at room temperature. Cells were embedded in 10% gelatin, infused in 2.3 M sucrose, and frozen in liquid nitrogen as described in detail previously (Raposo et al., 1997). Ultrathin cryosections were prepared with a Leica FCS ultracryomicrotome (Leica) and single and double immunogold labeled with a mouse monoclonal anti-Rab27A antibody and a rabbit polyclonal antichromogranin A/B according to Slot et al. (1991). A rabbit antimouse IgG was used after the first incubation with the monoclonal anti-Rab27A antibody. Protein Agold conjugates (PAG 10 and PAG 15) were purchased from the Department of Cell Biology, Utrecht University, Utrecht, Netherlands. Relative quantitation of the immunogold labeling for Rab27A was performed directly under the electron microscope by counting 650 gold particles associated with the different subcellular compartments of PC12 cells. 560 granules were analyzed for the presence of Rab27A.
Interaction assays
GST and GST-MyRIP constructs were expressed in E. coli BL21 cells and purified by affinity chromatography onto glutathioneSepharose (Amersham Biosciences) using standard procedures.
Pull Down.
Myosin-VIIa tail (El Amraoui et al. 2002) was expressed in COS-7 cells. Transfected COS-7 cells or PC12 cells were scrapped, sonicated, and solubilized at 4°C for 30 min in a buffer containing 1% Triton X-100, 150 mM NaCl, 25 mM Tris-Cl, pH 7.5, 2 mM ATP, and a cocktail of proteases inhibitors. After centrifugation (12,000 g, 15 min), supernatants (0.51 mg protein) were incubated for 1 h at 4°C under agitation with 1 nmol of purified GST or GSTMyRIP constructs prebound to 150 µl glutathioneSepharose beads. The beads were washed three times in 150 mM NaCl, 25 mM Tris-Cl, pH 7.5, and eluted in Laemmli sample buffer. Starting material and eluates were analyzed by SDS-PAGE and immunoblotting for the presence of myosin-Va or -VIIa.
Coimmunoprecipitation.
COS-7 cells were transfected 2 d before the experiment with vectors encoding myc-tagged MyRIP or MyRIP-Cter (1665). Cells were lysed as described above. Extracts were incubated with protein ASepharose beads (100 µl wet volume; Amersham Biosciences) conjugated with or without affinity-purified anti-MyRIP antibody. After three washes, the eluates were analyzed by SDS-PAGE and immunoblotting for the presence of MyRIP (with an antic-myc tag antibody) or actin.
Secretion assays
The [3H]5-HT release assay has been described previously (Schonn et al., 2003). In brief, PC12 cells were transfected with the serotonin transporter SERT, loaded with [3H]5-HT (520 Ci/mmol; Amersham Biosciences). Basal release was measured in Locke's solution containing (in mM) NaCl 154, KCl 5.5, glucose 5.6, NaHCO3 3.5, CaCl2 2.5, MgCl2 1.2, Hepes 15, and 0.2% BSA, pH 7.4, adjusted with NaOH supplemented with 1 µM clomipramine. Secretion was measured at 37°C in high K+ Locke's solution (55 mM K+ with Na+ reduced to 104.5 mM) in the presence of 1 µM clomipramine. After 210 min, the radioactivity released in the medium and remaining in the cells was measured. Alternatively, cells were rinsed twice with Ca2+-free Locke's solution and permeabilized for 4 min at 37°C with 20 µM digitonin or 22 U streptolysin-O in (in mM) potassium glutamate 139, Pipes 20, HEDTA 2, EGTA 2, free Mg2+ 1, ATP 2, plus 0.3% BSA and pH adjusted to 7 using KOH. Secretion was then elicited in the same medium supplemented with 30 µM free Ca2+, for 5 min at 37°C. Free Ca2+ concentrations were calculated according to Föhr et al. (1993) using calcv.22 software. Where indicated, cells were treated with 1 µM jasplakinolide or 5 µM latrunculin B for 20 min before being stimulated to secrete (in the continuing presence of the drug). The hGH release assay and amperometric recordings were performed as described previously (Schonn et al., 2003).
EW-FM
An upright microscope (Olympus BX50WI) has been adapted to EW-FM by the prism approach (Axelrod, 1981). To excite fluorescence, light from an argon laser set at 488 nm (Spectra Physics 177-G02) entered radially a hemispheric BK7 glass prism and struck its planar face at a supercritical angle. The angle was adjustable by means of telecentric optics to be described elsewhere, and the evanescent field decay length was calculated assuming a refractive index of 1.37 for the cellular medium (experimentally determined). NPYGFP-transfected PC12 cells cultured on polylysine-coated glass coverslips were observed in Locke's solution. Coverslips were optically coupled to the planar face of the prism with immersion oil (Carl Zeiss MicroImaging, Inc.). The laser intensity was attenuated to 1 mW, and illumination was restricted to image acquisition by a shutter coupled to the camera. The cells were observed through a water immersion objective 60X 0.9 NA (Olympus), and the images were captured with a CDD camera (Photometrics CoolSnap HQ; Roper Scientific). In the present work, frames were acquired at 2 Hz in stacks of 120 images. Exposure times were of 100300 ms, depending upon the signal to noise ratio, and under these conditions, <5% photobleaching occurred.
Analysis of SG motion
The positions of SGs in an x,y plane parallel to the membrane glass interface were determined using software modules provided by Metamorph (Universal Imaging). Occasionally, the image stacks were processed by a long pass filter (Steyer and Almers, 1999) before tracking the vesicles. For tracking, SGs that did not collide with neighboring granules and that stay bright for at least 30 images (15 s) were selected. A threshold brightness was then chosen, resulting in binary stacks where SGs appeared as single continuous regions of bright pixels. The position of the tracked SGs was determined as the mass center of these contiguous bright pixels. The trajectories were derived from the SG positions on each frame. For each SG trajectory, the MSD in the x,y plane was calculated according to Steyer and Almers (1999). Plots of MSD as a function of t were linear for
t < 5 s, and a two-dimension diffusion constant Dx,y was derived from the slope of the curve.
Statistical analyses
Significance of differences was calculated with t test or Mann-Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Online supplemental material
The supplemental material (Figs. S1S4, Videos 15, and supplemental Materials and methods) is available at http://www.jcb.org/cgi/content/full/jcb.200302157/DC1. Fig. S1 shows the presence of Rab27A in subcellular fractions enriched in SGs. Fig. S2 shows the distribution of Rab27A constructs in PC12 cells. Fig. S3 illustrates the cellular expression of the different constructs used in this study. Fig. S4 depicts how Rab27A and MyRIP may bridge SGs to actin. The videos illustrate the effect of Rab27A and MyRIP on SG motion. Plasmid constructions and protocols used for immunocytochemistry and subcellular fractionation are described in the supplemental Materials and methods.
![]() |
Acknowledgments |
---|
J.-S. Schonn was supported by a fellowship from the Fondation de la Recherche Médicale, and S. Huet was supported by the Direction Générale de l'Armement. This work was supported by a grant from the Ministère de la Recherche (DRAB).
Submitted: 25 February 2003
Accepted: 4 September 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Axelrod, D. 1981. Cellsubstrate contacts illuminated by total internal reflection fluorescence. J. Cell Biol. 89:141145.[Abstract]
Bahadoran, P., E. Aberdam, F. Mantoux, R. Busca, K. Bille, N. Yalman, G. de Saint-Basile, R. Casaroli-Marano, J.P. Ortonne, and R. Ballotti. 2001. Rab27a: a key to melanosome transport in human melanocytes. J. Cell Biol. 152:843850.
Barral, D.C., J.S. Ramalho, R. Anders, A.N. Hume, H.J. Knapton, T. Tolmachova, L.M. Collinson, D. Goulding, K.S. Authi, and M.C. Seabra. 2002. Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110:247257.
Bhakdi, S., U. Weller, I. Walev, E. Martin, D. Jonas, and M. Palmer. 1993. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med. Microbiol. Immunol. (Berl.). 182:167175.[Medline]
Darchen, F., A. Zahraoui, F. Hammel, M.P. Monteils, A. Tavitian, and D. Scherman. 1990. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules. Proc. Natl. Acad. Sci. USA. 87:56925696.[Abstract]
Denzer, K., M.J. Kleijmeer, H.F. Heijnen, W. Stoorvogel, and H.J. Geuze. 2000. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 113(Pt 19):33653374.
El-Amraoui, A., J.S. Schonn, P. Kussel-Andermann, S. Blanchard, C. Desnos, J.P. Henry, U. Wolfrum, F. Darchen, and C. Petit. 2002. MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes. EMBO Rep. 3:463470.
Föhr, K.J., W. Warchol, and M. Gratzl. 1993. Calculation and control of free divalent cations in solutions used for membrane fusion studies. Methods Enzymol. 221:149157.[Medline]
Fukuda, M., and T.S. Kuroda. 2002. Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin. J. Biol. Chem. 277:4309643103.
Fukuda, M., E. Kanno, C. Saegusa, Y. Ogata, and T.S. Kuroda. 2002a. Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells. J. Biol. Chem. 277:3967339678.
Fukuda, M., T.S. Kuroda, and K. Mikoshiba. 2002b. Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J. Biol. Chem. 277:1243212436.
Haddad, E.K., X. Wu, J.A. Hammer III, and P.A. Henkart. 2001. Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice. J. Cell Biol. 152:835842.
Hume, A.N., L.M. Collinson, A. Rapak, A.Q. Gomes, C.R. Hopkins, and M.C. Seabra. 2001. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J. Cell Biol. 152:795808.
Hume, A.N., L.M. Collinson, C.R. Hopkins, M. Strom, D.C. Barral, G. Bossi, G.M. Griffiths, and M.C. Seabra. 2002. The leaden gene product is required with Rab27a to recruit myosin Va to melanosomes in melanocytes. Traffic. 3:193202.[CrossRef][Medline]
Johannes, L., P.M. Lledo, M. Roa, J.D. Vincent, J.P. Henry, and F. Darchen. 1994. The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells. EMBO J. 13:20292037.[Abstract]
Lang, T., I. Wacker, I. Wunderlich, A. Rohrbach, G. Giese, T. Soldati, and W. Almers. 2000. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells. Biophys. J. 78:28632877.
Matesic, L.E., R. Yip, A.E. Reuss, D.A. Swing, T.N. O'Sullivan, C.F. Fletcher, N.G. Copeland, and N.A. Jenkins. 2001. Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defects observed in leaden mice. Proc. Natl. Acad. Sci. USA. 98:1023810243.
Ménasché, G., E. Pastural, J. Feldmann, S. Certain, F. Ersoy, S. Dupuis, N. Wulffraat, D. Bianchi, A. Fischer, F. Le Deist, and G. de Saint Basile. 2000. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat. Genet. 25:173176.[CrossRef][Medline]
Ménasché, G., J. Feldmann, A. Houdusse, C. Desaymard, A. Fischer, B. Goud, and G. de Saint Basile. 2003. Biochemical and functional charaterization of Rab27a mutations occurring in Griscelli syndrome patients. Blood. 101:27362742.
Nakata, T., and N. Hirokawa. 1992. Organization of cortical cytoskeleton of cultured chromaffin cells and involvement in secretion as revealed by quick-freeze, deep-etching, and double-label immunoelectron microscopy. J. Neurosci. 12:21862197.[Abstract]
Oheim, M., and W. Stuhmer. 2000. Tracking chromaffin granules on their way through the actin cortex. Eur. Biophys. J. 29:6789.[CrossRef][Medline]
Ornberg, R.L., L.T. Duong, and H.B. Pollard. 1986. Intragranular vesicles: new organelles in the secretory granules of adrenal chromaffin cells. Cell Tissue Res. 245:547553.[Medline]
Provance, D.W., Jr., M. Wei, V. Ipe, and J.A. Mercer. 1996. Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution. Proc. Natl. Acad. Sci. USA. 93:1455414558.
Raposo, G., M.J. Kleijmeer, G. Posthuma, J.W. Slot, and H.J. Geuze. 1997. Immunogold labeling of ultrathin cryosections: application in immunology. Handbook of Experimental Immunology. 5th ed. Vol. 4. L.A. Herzenberg, D. Weir, C. Blackwell, editors. Blackwell Science, Cambridge, MA. 111.
Rosé, S.D., T. Lejen, L. Casaletti, R.E. Larson, T.D. Pene, and J.M. Trifaro. 2003. Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion. J. Neurochem. 85:287298.[CrossRef][Medline]
Rudolf, R., T. Salm, A. Rustom, and H.H. Gerdes. 2001. Dynamics of immature secretory granules: role of cytoskeletal elements during transport, cortical restriction, and F-actin-dependent tethering. Mol. Biol. Cell. 12:13531365.
Rudolf, R., T. Kogel, S.A. Kuznetsov, T. Salm, O. Schlicker, A. Hellwig, J.A. Hammer III, and H.H. Gerdes. 2003. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells. J. Cell Sci. 116:13391348.
Sagné, C., C. Agulhon, P. Ravassard, M. Darmon, M. Hamon, S. El Mestikawy, B. Gasnier, and B. Giros. 2001. Identification and characterization of a lysosomal transporter for small neutral amino acids. Proc. Natl. Acad. Sci. USA. 98:72067211.
Schlüter, O.M., M. Khvotchev, R. Jahn, and T.C. Sudhof . 2002. Localization versus function of Rab3 proteins. Evidence for a common regulatory role in controlling fusion. J. Biol. Chem. 277:4091940929.
Schonn, J.S., C. Desnos, J.P. Henry, and F. Darchen. 2003. Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters. J. Neurochem. 84:669677.[CrossRef][Medline]
Slot, J.W., H.J. Geuze, S. Gigengack, G.E. Lienhard, and D. James. 1991. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J. Cell Biol. 113:123135.[Abstract]
Sönnichsen, B., S. De Renzis, E. Nielsen, J. Rietdorf, and M. Zerial. 2000. Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol. 149:901914.
Spector, I., N.R. Shochet, Y. Kashman, and A. Groweiss. 1983. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science. 219:493495.[Medline]
Steyer, J.A., and W. Almers. 1999. Tracking single secretory granules in live chromaffin cells by evanescent-field fluorescence microscopy. Biophys. J. 76:22622271.
Stinchcombe, J.C., D.C. Barral, E.H. Mules, S. Booth, A.N. Hume, L.M. Machesky, M.C. Seabra, and G.M. Griffiths. 2001. Rab27a is required for regulated secretion in cytotoxic T lymphocytes. J. Cell Biol. 152:825834.
Strobel, M.C., P.K. Seperack, N.G. Copeland, and N.A. Jenkins. 1990. Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles. Mol. Cell. Biol. 10:501509.[Medline]
Strom, M., A.N. Hume, A.K. Tarafder, E. Barkagianni, and M.C. Seabra. 2002. A family of Rab27-binding proteins: melanophilin links Rab27a and myosin Va function in melanosome transport. J. Biol. Chem. 277:2542325430.
Trifaro, J., S.D. Rose, T. Lejen, and A. Elzagallaai. 2000. Two pathways control chromaffin cell cortical F-actin dynamics during exocytosis. Biochimie. 82:339352.[CrossRef][Medline]
Wick, P.F., R.A. Senter, L.A. Parsels, M.D. Uhler, and R.W. Holz. 1993. Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells. J. Biol. Chem. 268:1098310989.
Wightman, R.M., J.A. Jankowski, R.T. Kennedy, K.T. Kawagoe, T.J. Schroeder, D.J. Leszczyszyn, J.A. Near, E.J. Diliberto, Jr., and O.H. Viveros. 1991. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. USA. 88:1075410758.[Abstract]
Wilson, S.M., R. Yip, D.A. Swing, T.N. O'Sullivan, Y. Zhang, E.K. Novak, R.T. Swank, L.B. Russell, N.G. Copeland, and N.A. Jenkins. 2000. A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proc. Natl. Acad. Sci. USA. 97:79337938.
Wu, X., B. Kocher, Q. Wei, and J.A. Hammer III. 1998. Myosin Va associates with microtubule-rich domains in both interphase and dividing cells. Cell Motil. Cytoskeleton. 40:286303.[CrossRef][Medline]
Wu, X., K. Rao, M.B. Bowers, N.G. Copeland, N.A. Jenkins, and J.A. Hammer III. 2001. Rab27a enables myosin Va-dependent melanosome capture by recruiting the myosin to the organelle. J. Cell Sci. 114:10911100.
Wu, X.S., K. Rao, H. Zhang, F. Wang, J.R. Sellers, L.E. Matesic, N.G. Copeland, N.A. Jenkins, and J.A. Hammer III. 2002. Identification of an organelle receptor for myosin-Va. Nat. Cell Biol. 4:271278.[CrossRef][Medline]
Yi, Z., H. Yokota, S. Torii, T. Aoki, M. Hosaka, S. Zhao, K. Takata, T. Takeuchi, and T. Izumi. 2002. The Rab27a/granuphilin complex regulates the exocytosis of insulin-containing dense-core granules. Mol. Cell. Biol. 22:18581867.
Zhao, S., S. Torii, H. Yokota-Hashimoto, T. Takeuchi, and T. Izumi. 2002. Involvement of Rab27b in the regulated secretion of pituitary hormones. Endocrinology. 143:18171824.